osteoarthritis ( oa ) is characterized by cartilage breakdown , synovial fibrosis , and osteophyte formation .
the main clinical symptom of oa is chronic joint pain , which is typically treated using analgesic drugs , such as nonsteroidal anti - inflammatory drugs and corticosteroids [ 1 , 2 ] .
however , due to potential adverse effects [ 3 , 4 ] and variable efficacy of these drugs for providing symptomatic pain relief , more effective analgesics are needed to improve oa treatment and patient care .
in addition , because only a few effective disease - modifying drugs are available for oa [ 5 , 6 ] , a better understanding of the mechanisms that drive oa pain is required to guide drug development . nerve growth factor ( ngf ) is a widely known pain mediator   and plays a critical role in the modulation of oa pain [ 811 ] .
the neutralization of ngf with tanezumab , an anti - ngf monoclonal antibody , has robust analgesic effects on oa pain [ 8 , 10 , 11 ] . due to these effects
, the regulation of ngf has been investigated in several in vitro and in vivo studies [ 12 , 13 ] .
evidence from these studies suggests that the activity of ngf is mediated by inflammatory cytokines .
for example , in an experimental arthritic mouse model , il-1 , but not tnf- , increased ngf levels in knee joints .
however , the mechanism regulating ngf expression in synovial tissue ( st ) remains unclear .
st contains macrophage- and fibroblast - like cells in the lining layer [ 1416 ] .
the treatment of cultured synovial oa fibroblasts with il-1 and tnf- induces the production of ngf .
more recent studies have shown that macrophages produce a number of inflammatory cytokines , including il-1 , il-6 , and tnf- , which contribute to oa progression and associated joint pain [ 1721 ] . despite these findings , the regulation of ngf expression in synovial macrophages is not fully understood .
str / ort mice are a well - characterized oa model that spontaneously develop oa with a progression resembling that of humans [ 2224 ] .
synovial hyperplasia is also observed in str / ort mice , and we previously showed that the cd11b+ macrophage population in st str / ort mice is higher than that found in c57bl/6j mice [ 17 , 18 ] .
in addition , we reported that compared to synovial fibroblasts ( cd11b ) , synovial macrophages produce high levels of il-1 and tnf- in str / ort mice . here
, we characterized the expression profiles of several inflammatory cytokines and ngf in the st of str / ort mice .
in addition , the regulation of ngf expression by inflammatory cytokines in synovial macrophages and fibroblasts was also examined .
nine - month - old male str / ort ( average body weight , 40.6  4.1  g ) and c57bl/6j ( control ; average body weight , 32.8  1.2  g ) mice ( charles river laboratories , inc . ,
specific pathogen - free colonies of each mouse line were maintained at nippon charles river laboratories ( kanagawa , japan ) in a semibarrier system with a controlled environment ( temperature : 23  2c ; humidity : 55%  10% ; lighting : 12  h light / dark cycle ) .
all experimental protocols were approved by the kitasato university school of medicine animal care committee . c57bl/6j and
str / ort mice were sacrificed by the intramuscular injection of a mixture of medetomidine , midazolam , and butorphanol tartrate . after removing skin with a scalpel ,
st was harvested , and total rna was then extracted from the harvested tissue using trizol ( invitrogen , carlsbad , ca , usa ) , according to the manufacturer 's instructions .
the extracted total rna was used as a template for first - strand cdna synthesis using superscript iii rt ( invitrogen ) in pcr reaction mixtures consisting of 2  l cdna , specific primer set ( 0.2  m final concentration ) , and 12.5  l sybr premix ex taq ( takara , kyoto , japan ) in a final volume of 25  l .
the primers for il6 and ngf were designed using primer blast software and were synthesized by hokkaido system science co. , ltd .
the other primers used in this study were designed based on previously published primer sequences .
the sequences of the pcr primer pairs used in this study are listed in table 1 .
quantitative pcr was performed using a cfx-96 real - time pcr detection system ( bio - rad , hercules , ca , usa ) .
the pcr cycles consisted of an initial denaturation step at 95c for 1  min , followed by 40 cycles of 95c for 5  s and 60c for 30  s. mrna expression was normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) mrna .
the gene expression levels in the st of str / ort mice were compared with those in the st of c57bl/6j mice ( str / ort / c57bl6j ) .
in addition , the gene expression levels in cd11b+ cells were compared with those in cd11b cells ( cd11b+/cd11b ) .
st samples were collected from both knees of five str / ort mice , and mononuclear cells were then isolated from the collected tissue by digestion with type i collagenase for 2  h at 37c .
the obtained mononuclear cells were suspended in 500  l phosphate - buffered saline ( pbs ) containing biotinylated anti - cd11b antibody . following a 30  min incubation at 4c , the cells were washed with pbs , mixed with streptavidin - labelled magnetic particles ( bd imag streptavidin particles plus - dm ; bd biosciences , tokyo , japan ) , and incubated for 30  min on ice in an imag separation system ( bd biosciences )
. warmed ( 37c ) -minimum essential medium ( mem ) was added to the cell suspension to collect unbound ( cd11b - negative ) cells , and an additional 3  ml -mem was added to collect cd11b - positive cells after removing the tub from the magnetic support .
the cd11b - positive and cd11b - negative cells were collected by centrifugation at 300  g for 10  min , and the obtained cells were cultured in -mem in six - well plates .
tnf- , il-1 , il-6 , and ngf expression in the cells was analyzed by reverse transcription - polymerase chain reaction ( rt  pcr ) .
st - derived mononuclear cells , including synovial macrophages and fibroblasts , which were isolated as described above , were cultured in -mem in six - well plates . after a 1-week incubation at 37c in a 5% co2 incubator
, synovial fibroblasts were incubated with either mouse recombinant tnf- ( 25  ng / ml ) , il-1 ( 50  ng / ml ) , or il-6 ( 100  ng / ml ) ( biolegend , san diego , ca , usa ) for 24  h. cells that were not treated with any cytokines were used as controls . to examine the effect of tnf- on synovial macrophages and fibroblasts ,
cd11b - positive and cd11b - negative cells were collected from st , as described above , and were then cultured in -mem in six - well plates . after a 1-week incubation at 37c in a 5% co2 incubator
, cd11b - positive and negative cells were incubated with mouse recombinant tnf- ( 25  ng / ml ) or il-1 ( 50  ng / ml ) for 24  h. cells that were not treated with tnf- were used as controls .
the treated and control cells were harvested for total rna isolation , as described above , and ngf expression was analyzed by rt  pcr .
ten l3 and l4 drg samples were harvested from the 9-month - old c57bl/6j and str / ort mice and were then immersed in a buffered paraformaldehyde fixative at 4c overnight .
the samples were further incubated in pbs containing 20% sucrose for 24  h at 4c . after freezing in liquid nitrogen ,
each specimen was sectioned at 10  m thickness on a cryostat ( leica microsystems , cm3050s , wetzlar , germany ) and was then treated for 90  min at room temperature with a blocking solution of pbs containing 0.05% tween-20 and 1% skim milk .
the sectioned drg specimens were stained with rabbit antibodies against calcitonin gene - related peptide ( cgrp ; 1  :  1000 ; immunostar , hudson , wi , usa ) and transient receptor potential vanilloid 1 ( trpv1 ; 1  :  500 ; calbiochem , san diego , ca , usa ) by incubation for 18  h at 4c .
the drg sections were then incubated with alexa 488-conjugated goat anti - rabbit igg ( for cgrp immunoreactivity , 1  :  1000 ; molecular probes ) and alexa 488-conjugated goat anti - rabbit igg ( for trpv1 immunoreactivity , 1  :  1000 ; molecular probes ) .
the immunostained sections were visualized using a fluorescence microscope ( axiovert 200 , zeiss , jena , germany ) in a treatment - blinded manner .
the numbers of cgrp- and trpv - positive cells were counted , and the proportion of these cells to the total number of nucleated cells in drg was calculated for each drg sample .
differences between c57bl/6j and str / ort mice were examined using the t - test .
all statistical analyses were performed with spss software ( version 11.0 ; spss , inc . ,
chicago , il , usa ) . a p value of < 0.05 was considered statistically significant .
real - time pcr analysis of the genes encoding tnf- , il-1 , il-6 , and ngf showed that the expression levels of these genes were significantly elevated in the st of str / ort mice compared to those in control c57bl/6j mice ( figures 1(a)1(d ) ) . activated
macrophages reportedly produce higher levels of inflammatory cytokines , including tnf- , il-1 , and il-6 , than tissue - resident macrophages [ 18 , 26 , 27 ] . to determine whether macrophages in st also produce inflammatory cytokines , expression of the genes encoding tnf- , il-1 , and il-6 in cd11b - positive cells isolated from the st of str / ort mice was examined by real - time pcr .
tnf- , il-1 , and il-6 gene expression in cd11b - positive cell fractions was higher than that in cd11b - negative cell fractions ( figures 2(a)2(c ) ) .
in contrast , no differences in the gene expression of ngf were detected between the two examined cell fractions ( figure 2(d ) ) .
real - time pcr analysis revealed that the gene expression of ngf increased significantly in isolated synovial cells in the presence of exogenously added tnf- and il-1 compared to untreated control cells ( figure 3 ) .
in contrast , the gene expression of ngf was not affected in il-6-treated synovial cells ( figure 3 ) .
ngf expression was also significantly increased in the presence of exogenously added tnf- in isolated populations of synovial fibroblasts and macrophages compared to untreated control cells ( figure 4(a ) ) .
ngf expression was also significantly increased in the presence of exogenously added il-1 in isolated populations of synovial fibroblasts , but not in synovial macrophages ( figure 4(b ) ) . to investigate pain - related sensory innervation by ngf , the proportion of trpv1- and cgrp - positive cells in the drg of str / ort mice was investigated by immunohistochemistry .
the number of trpv1- ( figures 5(a ) , 5(b ) , and 5(e ) ) and cgrp - positive cells ( figures 5(b ) , 5(d ) , and 5(f ) ) in the drg of str / ort mice was significantly higher compared to that found in the drg of c57bl/6j mice .
in the present study investigating the mechanisms underlying the regulation of ngf and development of oa pain , higher expression of the genes encoding tnf- , il-1 , il-6 , and ngf was observed in the st of an oa str / ort mouse model compared to that in c57bl/6j mice .
in addition , ngf expression was specifically detected in the cd11b - positive and cd11b - negative cell fractions isolated from st , whereas higher tnf- , il-1 , and il-6 gene expression was observed in the cd11b - positive cell fractions .
notably , the treatment of cultured synovial cells with tnf- and il-1 stimulated ngf expression and tnf- also stimulated ngf expression in cd11b - positive and cd11b - negative cell fractions .
the number of trpv1- and cgrp - positive cells in the drg of str / ort mice was significantly higher compared to that found in the drg of c57bl/6j mice .
taken together , these findings suggest that tnf- regulates ngf expression in both synovial fibroblasts and macrophages and that il-1 regulates ngf expression in synovial fibroblasts and that these factors may contribute to the development of pain in oa patients by innervation of the peripheral nervous system .
ngf is upregulated in human synovial fibroblasts , suggesting that this factor plays an important role in oa pathology [ 12 , 13 , 28 ] .
ngf elevated inflammatory knee joint and tnf- and il-1 stimulate synovial fibroblasts to produce ngf .
reported that ngf expression is upregulated in the knee joint of a carrageenan - treated inflammatory arthritis model and is further stimulated by the intraarticular injection of tnf- and il-1 .
further , tnf- and il-1 treatment of cultured synovial fibroblasts derived from a human oa patient also increased ngf production .
however , because these studies consisted of analyses of whole knee joints or cultured synovial fibroblasts exogenously stimulated with tnf- and il-1 , the cell populations responsible for regulating ngf and producing tnf- and il-1 in st were not conclusively determined . here ,
elevated expression of the inflammatory cytokines tnf- , il-1 , and il-6 was observed in the st of str / ort mice , and tnf- and il-1 were increased in synovial macrophages isolated from this oa model . in addition , ngf expression was stimulated by tnf- and il-1 in the synovial fibroblast fraction .
taken together , these findings suggest that tnf- and il-1 stimulate ngf expression in synovial fibroblasts .
a recent immunohistochemical analysis revealed that ngf not only is localized to fibroblasts , but is also produced by a specific population of macrophages in human st .
however , the factor regulating ngf in macrophages was not determined . in the present study ,
isolated synovial macrophages and fibroblasts from str / ort mice expressed ngf at similar levels to each other .
ngf expression in the macrophage fraction was also stimulated by tnf-. taken together , these findings indicate that tnf- stimulates ngf expression in both synovial fibroblasts and macrophages .
ngf plays a key role in the generation of acute and chronic pain and peripheral sensitization [ 3033 ] .
recent evidence suggests that the trpv1 ion channel and cgrp are involved in the development of ngf - induced persistent mechanical and thermal hypersensitivity in rats [ 30 , 31 ] .
ngf acutely modulates trpv1 activity [ 32 , 33 ] and also stimulates cgrp expression in drg neurons in vitro and in vivo .
consistent with this speculation , inhibition of ngf by treatment with tanezumab was recently shown to have high analgesic efficacy in oa knees compared with placebo . in the present study , cgrp- and trpv1-positive cells , which are localized in drg neurons in monoiodoacetate- ( mia- ) treated oa animals [ 35 , 36 ] ,
were increased in the drg of str / ort compared to c57bl/6j mice , suggesting that the elevation of synovial ngf in str / ort mice contributes to peripheral sensitization .
however , several studies have shown that human oa chondrocytes also produce ngf by inflammatory cytokines [ 37 , 38 ] .
further investigations are needed to clarify the contribution of chondrocytes derived ngf on peripheral sensitization .
first , the mechanism underlying the development of oa pain that was proposed based on the present results was based on cross - sectional analysis and therefore no data on the effects of the observed changes on the progression of oa are available .
second , although ngf was found to be elevated in the st of oa mice , it remains to be determined if the elevation of synovial ngf levels contributes to peripheral sensitization .
finally , present study did not confirm that the protein levels of these cytokines correlated with the expression levels . in conclusion , the present results support the hypothesis that tnf- regulates ngf expression in synovial fibroblasts and macrophages and il-1 regulates ngf expression in synovial fibroblasts in oa mice .
tnf- and il-1 may therefore regulate ngf signaling in oa joints and be suitable therapeutic targets for treating oa pain .
 to investigate the role of macrophages as a regulator and producer of nerve growth factor ( ngf ) in the synovial tissue ( st ) of osteoarthritis ( oa ) joints , the gene expression profiles of several inflammatory cytokines in the st , including synovial macrophages and fibroblasts , of oa mice ( str / ort ) were characterized . 
 specifically , real - time polymerase chain reaction analysis was used to evaluate the expression of tumor necrosis factor- ( tnf- )  , interleukin- ( il- ) 1 , il-6 , and ngf in cd11b+ and cd11b cells isolated from the st of a murine oa model . 
 the effects of tnf- , il-1 , and il-6 on the expression of ngf in cultured synovial cells were also examined . 
 the expression of tnf- , il-1 , il-6 , and ngf in the st of str / ort was higher than that in c57/bl6j mice . 
 compared to the cd11b cell fraction , higher expression levels of tnf- , il-1 , and il-6 were detected in the cd11b+ cell fraction , whereas no differences in the expression of ngf were detected between the two cell fractions . 
 notably , tnf- upregulated ngf expression in synovial fibroblasts and macrophages and il-1 upregulated ngf expression in synovial fibroblasts . il-1 and tnf- may regulate ngf signaling in oa joints and be suitable therapeutic targets for treating oa pain . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion