rheumatoid arthritis ( ra ) is a systemic 
 autoimmune disease characterized by the progressive 
 chronic inflammation of 
 multiple joints . the incidence of ra is close to 1% worldwide , 
 but its etiology 
 is not yet known . the search for therapeutic agents for ra is being conducted mainly 
 using collagen - induced arthritis and adjuvant - induced arthritis 
 [ 1 , 2 ] . in the 
 development of arthritis , pannus formation is the 
 critical step in the 
 destruction of cartilage and bones . pannus is a 
 proliferative granulomatous tissue , 
  mainly 
 composed of macrophages and fibroblast - like synoviocytes . although collagen - induced arthritis and 
 adjuvant - induced arthritis models are very 
 useful for screening candidates for 
 novel antirheumatic drugs , it is not easy to elucidate 
 the effects of 
 candidates on the pannus formation . also , quantitative 
 analysis of cytokine 
 levels is required to elucidate the role of cytokines 
 in the pathogenesis 
 of pannus formation . however , many steps are needed 
 to measure cytokines in the 
 pannus in arthritis models . for example , the ankle 
 joints have to be removed , 
 frozen in liquid nitrogen , crushed , homogenated , 
 and finally cytokines are 
 assayed by enzyme - linked immunosorbent assay ( elisa ) 
 in the supernatants . therefore , 
 an inflammatory model is needed to simply evaluate the 
 effects of drugs on 
 pannus formation and cytokine production in pannus . there is only a few animal models available for 
 the evaluation 
 of antiarthritic drugs on pannus 
 formation . one of the granuloma models , a cotton 
 pellet - induced granuloma model , 
 is widely used as a model for pannus formation 
 . the problem with this model is , however , 
 that the granuloma is formed by a 
 foreign - body - dependent granuloma reaction . the mechanism of pannus formation 
 has not been fully proven yet , 
 but t cells seem to be involved in pannus 
 formation . 
 another granuloma model , delayed - type hypersensitivity ( dth ) 
 granuloma , has been reported to be antigen - specific 
 and t cell - mediated [ 68 ] . histological features of the lesions from 
 dth granuloma revealed the evolution 
 of dth into a chronic glaucomatous reaction , as 
 shown by the following changes : 
 fibrin deposition , vasculitis , mononuclear cell 
 infiltration / proliferation , and 
 angiogenesis . they reported that dth granuloma represented a relevant model 
 for probing pathogenic mechanisms and potential 
 therapeutics for ra . in this 
 study , we modified the dth granuloma 
 model so that we could easily measure 
 cytokine production from the granulomatous 
 tissue . the granuloma was induced by 
 a subcutaneous implantation of a thin 
 membrane filter soaked in methylated bovine 
 serum albumin ( mbsa ) solution into 
 the back of the mice previously sensitized 
 with mbsa . for cytokine determination , 
 the granuloma was collected from the back of 
 the mice on the indicated days and 
 incubated in culture medium for 24 hours in vitro , 
 and the cytokines were measured 
 in the supernatants . phosphodiesterase 4 ( pde4 ) inhibitors are key 
 enzymes that degrade camp and play an important 
 role in inflammatory and immune 
 reactions . the prevention of camp degradation by pde4 
 inhibitors elevates 
 the level of camp in the cells followed 
 by suppression of inflammation and 
 immune responses . rolipram is a well - known 
 pde4 inhibitor that reduces the 
 inflammatory responses in several rodent models , 
 including carrageenan - induced 
 edema , 
 adjuvant arthritis [ 10 , 11 ] , 
 antigen - induced airway inflammation 
 [ 12 , 13 ] , 
 lipopolysaccharide- ( lps- ) induced lung 
 inflammation   and 
 collagen - induced arthritis 
 . inhibitions of leukotriene b4 
 ( ltb4 ) , 
 interferon- ( ifn- ) , tumor necrosis 
 factor- ( tnf- ) , 
 interleukin-4 ( il-4 ) , 
 and il-5 are all potential mechanisms 
 by which the anti - inflammatory effects of 
 rolipram are mediated . these results suggest that pde4 inhibitors may be 
 therapeutic agents for various diseases such 
 as asthma , chronic obstructive 
 pulmonary disease , and rheumatoid arthritis . 
 in this study , we evaluated pde4 
 inhibitors , kf66490 and sb207499 
 , 
 in the mouse pannus model and compared 
 the effects of the pde4 inhibitors with 
 those of typical antirheumatic drugs 
 leflunomide , prednisolone , and methotrexate . male , 6-week - old balb / c mice were purchased 
 from charles river japan ( kanagawa , japan ) 
 and maintained at a 
 temperature of 223c 
 and a humidity of 5020% . the study protocol for the animal 
 experiment was approved 
 by the animal care committee of kyowa hakko 
 kogyo co. , ltd . prednisolone , diclofenac sodium and methylated 
 bovine serum albumin ( mbsa ) were purchased 
 from sigma - aldrich japan 
 ( tokyo , japan ) . sources of other chemicals 
 were as follows : freund 's complete adjuvant 
 ( difco , detroit , mich , usa ) ; myeloperoxidase 
 ( sigma - aldrich japan ) , hexadecyltrimethylammonium 
 bromide ( sigma - aldrich japan ) ; elisa 
 kits for each mouse cytokine : il-6 
 and il-1 ( biosource 
 international , camarillo , calif , usa ) , tnf- 
 ( r&d systems , minneapolis , mn , usa ) , ifn- ( pierce , 
 rockford , ill , usa ) , il-4 
 ( bd bioscience , san diego , 
 calif , usa ) , il-10 ( bd bioscience ) , 
 il-12 ( genzyme , cambridge , mass , usa ) ; 
 rpmi 1640 tissue culture medium 
 ( sigma - aldrich ) ; fetal bovine 
 serum ( intergen , purchase , ny , usa ) ; 
 penicillin - streptomycin ( life technologies , 
 rockville , md , usa ) ; and 2-mercaptoethanol 
 ( wako pure chemical ) . granuloma was induced according to dunn et al . 
 
 with a slight modification . methylated 
 bsa ( mbsa : 4  mg / ml in saline ) was emulsified in 
 an equal volume of freund 's 
 complete adjuvant . the emulsion 
 ( 0.1  ml containing 200  g 
 mbsa ) was injected intradermally on 
 the back of the mice . two weeks 
 after the last sensitization , a membrane 
 filter ( 13  mm diameter , 0.05  mm thickness , 
 millipore japan , tokyo , japan ) which 
 had been soaked in mbsa solution 
 ( 4  mg / ml in saline ) for 60 minutes was subcutaneously 
 implanted in the dorsum of the mice ( day 0 ) . the wound was closed with 9  mm autoclips 
 ( clay adams , parsippany , nj ) . the granuloma formed around the filter was collected from the 
 back of the mice and chopped into pieces 
 in 4  ml of 1640 tissue culture 
 medium supplemented with 
 10% heat - inactivated fetal bovine serum ( fbs ) , 
 50  unit / ml penicillin-50  g / ml streptomycin , 
 and 50  mol / l 2-mercaptoethanol . anti - mbsa specific 
 igg1 and 
 igm antibodies in the 
 supernatants were measured by elisa 
 , 
 and the results were expressed as 
 the absorbance at 450  nm . myeloperoxidase activity was measured 
 using a specific substrate in the mixture of the 
 culture medium and the granulomatous tissue . briefly , 
 the chopped tissues and the remaining supernatants 
 ( total 3.2  ml ) were mixed with 
 0.16  ml of potassium phosphate buffer 
 ( 0.5  mol / l , ph 6.0 ) and 0.16  ml 
 hexadecyltrimethylammonium 
 bromide solution ( 10% ) and vigorously vortexed . after 
 centrifugation ( 1800  g , 20 minutes ) , 
 the enzyme activity in the supernatant was 
 measured . 
 the remaining tissue and the supernatant were mixed with an 
 equal amount ( 3.3  ml ) of hcl ( 12  mol / l ) 
 and incubated for 15 hours at 110c for 
 the measurement of hydroxyproline content 
 according to the procedure of jakubzick 
 . data 
 were expressed as total enzyme activity 
 or total hydroxyproline content in the granuloma . the granuloma was collected on days 1 , 3 , 
 6 , 10 , 15 , 22 , and 30 and cultured in vitro . a saline - soaked filter was 
 implanted in the back of the mice as a 
 negative control group ( saline group ) . all drugs were suspended in 0.5% methylcellulose 
 in distilled water and orally administered 
 once a day from day 0 to 10 . male balb / c mice ( 810 weeks old ) were injected 
 intraperitoneally with 1  ml of 3% ( w / vol ) 
 thioglycolate broth ( wako pure chemical ) . four days later , cells were harvested by 
 peritoneal lavage with rpmi 1640 
 medium . the cells were washed twice , and 
 2.5  104 
 peritoneal cells ( for 
 tnf- production ) or 2  105 
 peritoneal cells ( for il-12 production ) per 0.2  ml in 
 the same medium containing 10% fbs were 
 then added to each well of 96-well plates . the cells were allowed to adhere to 
 well at 37c 
 in a humidified 5% co2 
 atmosphere , and after 2 hours , nonadherent 
 cells were removed by gentle washing . adherent cells were 
 stimulated with 1  g / ml 
 of lps for 6.5 hours ( for tnf- ) or 24 hours ( for 
 il-12 ) at 37c . surface parts of synovial tissues were isolated from knee 
 joints of female , 8-week - old lewis rats 
 ( charles river japan ) , followed by washing 
 with phosphate - buffered saline ( pbs ) and subsequent 
 digestion with 0.2% 
 collagenase in rpmi 1640 medium at 37c for 2 hours . synovial 
 tissues were then treated with 0.2% 
 collagenase and 0.25% trypsin at 37c 
 for 2 hours . cells were collected in rpmi 
 1640 containing 10% fbs and 
 antibiotics , and then centrifuged at 500  xg for 5 minutes . synovial fibroblasts ( 1105 
 cells ) suspended in 200  l of rpmi 1640 medium 
 supplemented with 1% fbs , 0.9% bsa and antibiotics were 
 precultured for 24 hours at 37c . after the preculture , the culture medium was changed 
 to rpmi 1640 medium supplemented 
 with 10% fbs and cells were cultured in the 
 presence or absence of compounds 
 for 48 hours at 37c followed by 24 hours 
 culture in the presence of 1.25  ci / ml 
 of [ methyl - h ] 
 thymidine . in the case of methotrexate and 
 leflunomide , 1.25  ci / ml of [ 6-h ] 
 deoxyuridine was also used instead 
 of [ methyl - h ] 
 thymidine [ 21 , 22 ] . the incorporation of [ h ] 
 thymidine or [ 6-h ] deoxyuridine 
 was counted by a liquid 
 scintillation counter . the student 's 
 t test or 
 aspin - welch test following an f - test was 
 used for analysis of differences between 
 the two groups . multiple comparisons among 
 treatment groups were performed by 
 1-way anova followed by the dunnett test or 
 kruskal - wallis followed by the steel 
 test . the granulomas collected on the indicated days 
 were chopped into pieces in 
 culture medium . cytokine concentrations and 
 anti - mbsa specific igg1 and 
 igm antibody titer in the supernatants were 
 measured after 24-hour incubation without 
 any manipulation to extract cytokines 
 from the granulomatous tissues 
 ( figure 1 ) . the concentration 
 of il-1 peaked on day 10 and then gradually decreased in 
 the mbsa - treated group 
 ( figure 1(a ) ) . tnf- and 
 il-6 were both released on day 1 , transiently 
 decreased on day 3 , and were constantly 
 released until day 30 ( figures 
 1(b ) , 1(c ) ) . il-12 release peaked on day 6 , while ifn- 
 was slightly delayed and peaked on day 10 
 ( figure 1(d ) and data not shown ) . il-4 and il-10 were significantly increased only 
 on day 1 ( data not shown ) . anti - mbsa specific 
 igg1 antibody concentration 
 was significantly increased from day 6 
 and sustained until day 22 ( data not 
 shown ) . anti - mbsa specific igm antibody 
 concentration was significantly 
 increased from day 1 and peaked on day 15 
 ( data not shown ) . myeloperoxidase 
 ( mpo ) activity , a marker enzyme for neutrophil 
 infiltration in the mixture of 
 the culture medium and the granuloma , 
 was constantly high in the mbsa - treated 
 group ( figure 1(e ) ) . hydroxyproline 
 content , a marker of fibrosis , increased with 
 time and peaked on day 15 in the 
 granuloma ( figure 1(f ) ) . drugs were orally administered for 10 
 days from the day of the implantation of mbsa - soaked filter in the back of the mice . il-1 
 production from the granuloma was suppressed 
 by both pde4 inhibitors , kf66490 
 and sb207499 , leflunomide , and prednisolone , 
 but not by methotrexate ( figure 2(a ) ) . il-6 production was suppressed by all drugs 
 except kf66490 ( figure 2(c ) ) . il-12 production was suppressed by 
 both pde4 inhibitors and prednisolone and partially 
 by leflunomide but not by methotrexate ( figure 2(d ) ) . increase in mpo activity 
 was suppressed by all drugs except for 
 methotrexate ( figure 2(e ) ) . increase in hydroxyproline 
 content was suppressed by all drugs except 
 methotrexate ( figure 2(f ) ) . pde4 inhibitors suppressed anti - mbsa specific 
 igg1 antibody production , 
 but not anti - mbsa specific igm antibody 
 production . leflunomide and methotrexate 
 strongly suppressed both 
 igg1 
 and igm antibody production . nonsteroidal anti - inflammatory drugs ( nsaids ) , 
 diclofenac significantly increased the tnf- 
 and il-12 production . none of the drugs 
 affected the body weight increase 
 except prednisolone , which significantly 
 suppressed it both at 10 and 30  mg / kg 
 ( data not shown ) . to evaluate the effects of pde4 inhibitors on proinflammatory 
 cytokine production , lps - stimulated tnf- 
 and il-12 production from thioglycolate - elicited 
 peritoneal macrophages were 
 conducted in vitro . both pde4 inhibitors 
 suppressed lps - stimulated tnf- and 
 il-12 production with ic50 
 values of 0.88 and 4.2 mol / l 
 ( kf66490 ) , and 0.12 and 0.52  mol / l 
 ( sb207499 ) , respectively ( table 3 ) . to evaluate the 
 antiproliferative activity of pde4 inhibitors and 
 antirheumatic drugs on 
 fibroblasts kf66490 
 and sb207499 inhibited serum - stimulated proliferation 
 of rat synovial 
 fibroblasts with an ic50 
 values of 9.2 and 3.1 we have shown that 
 this pannus model makes it much easier to 
 determine a variety of cytokines produced 
 in the granuloma than the previous methods 
 which needs many steps to extract 
 cytokines from tissue , and the type of 
 cytokines is very similar to that 
 reported to be increased in the pannus 
 in the patients with ra . these 
 results suggest that this model could 
 be a relevant model for evaluation of 
 pannus formation in patients with ra . 
 both pde4 inhibitors , kf66490 and sb207499 , 
 showed an effect comparable to those of 
 antirheumatic drugs in this model ; and 
 also , pde4 inhibitors inhibited 
 lipopolysaccharide - induced tnf- and il-12 
 production from thioglycolate - induced 
 murine peritoneal macrophages and the 
 proliferation of rat synovial 
 fibroblasts . therefore , pde4 inhibitors 
 could have therapeutic effects on 
 pannus formation in ra by inhibition 
 of cytokine production by 
 macrophages and synovial fibroblast proliferation . there was no report on the measurement 
 of cytokines in delayed - type hypersensitivity 
 granuloma . we compared cytokine concentrations 
 between in - tissue homogenate samples and 
 in - culture 
 supernatants of the granuloma . however , a marked variability of cytokine 
 concentrations was observed in 
 the case of using homogenate samples ( data not 
 shown ) . the new method showed that 
 the variation was relatively small and could 
 be useful for the measurment of 
 cytokine productions from granulomatous tissues . a broad array of macrophages and fibroblast 
 cytokines , including tnf- , il-1 , il-6 , and il-12 are 
 produced by the rheumatoid 
 synovium in the pannus [ 2325 ] . tnf- , il-1 , 
 and il-6 have been 
 reported to play a pivotal role in the 
 pathogenic mechanisms of ra [ 2628 ] . in this study , chronic production of tnf- , 
 il-1 , il-6 , and il-12 was observed . these observations suggest that this pannus 
 model is useful for evaluating the 
 effects of drugs on the production of 
 cytokines involved in the pathogenic mechanisms 
 of ra . pde4 inhibitors suppressed these 
 all cytokine production in vivo and 
 tnf- and il-12 production in vitro . these results suggest that pde4 inhibitors 
 directly suppress the production of tnf- 
 and il-12 , and indirectly suppress the 
 production of il-1 and il-6 which are possibly induced 
 by tnf- stimulation . because it has been 
 reported that tnf- transgenic mice 
 develop arthritis with enhanced production 
 of tnf- , il-1 , and il-6 
 , which 
 means tnf- is the major dominant regulator of il-1 
 and il-6 . additional explanation of antipannus forming effects of pde4 
 inhibitors is the inhibition of 
 fibroblast proliferation . mol / l 
 at the maximum dose used in this 
 study ( 30  mg / kg p.o . ) 
 . therefore , the inhibition of fibroblast 
 proliferation is considered to be involved 
 in antipannus forming effects of 
 pde4 inhibitors . some autoantibody 
 levels in the synovial fluid are specifically 
 related to the pathogenesis of ra 
 [ 32 , 33 ] . this finding 
 conflicts with previous reports that pde4 
 inhibitors inhibit igg1 or 
 igg2a antibody production in 
 vivo [ 34 , 35 ] , 
 but they did not measure the antibody levels 
 in the inflammatory 
 sites . these results suggest that 
 pde4 inhibitors inhibit antibody production 
 in the inflammatory sites . it is 
 not known exactly why pde4 inhibitors 
 suppressed the igg1 
 production ; but in our model , il-4 production on 
 day 1 was completely inhibited by both pde4 
 inhibitors ( 85 or 83% inhibition at 
 10 or 30  mg / kg of kf66490 and 53 or 
 103% inhibition at 10 or 30  mg / kg of 
 sb207499 ) . il-4 is known to promote the 
 proliferation and differentiation of 
 activated b - cells . therefore , the 
 suppression of il-4 production by pde4 
 inhibitors may have had some inhibitory effects on b cells . it has been reported that pde4 inhibitors 
 suppress both th1 and 
 th2 cytokine 
 production from stimulated t cells in vitro 
 [ 36 , 37 ] . however , the inhibition 
 is partial . therefore , we believe 
 that the suppression of t cell function is less 
 involved in antipannus effects of 
 pde4 inhibitors because no obvious inhibition 
 of ifn- production was 
 observed in our model ( data not shown ) ; 
 and also , we did not find any effect on ifn- 
 and il-6 production from concanavalin . 
 a stimulated murine splenic t cells up to 10  mol / l 
 ( data not shown ) . interestingly , 
 it has been reported that dendritic cells 
 exposed to pde4 inhibitors during 
 maturation reduce the development of ifn--expressing 
 effector t cells . therefore , pde4 inhibitors seem to affect the 
 development of th1 
 cells in vitro ; but as mentioned above , il-4 production was 
 completely inhibited by pde4 
 inhibitors in our model . the suppression 
 of il-4 production by pde4 inhibitors 
 might have had an impact on 
 th1/th2 
 balance . taken 
 together , although pde4 inhibitors preferentially 
 diminish th1 
 responses , some 
 effects of pde4 inhibitors on th2 
 responses [ 36 , 37 ] might complicate 
 the in vivo responses . consequently , 
 we think the effects of pde4 inhibitors on 
 in vivo th1/th2 
 responses depend on the type of 
 inflammation . recently , il-17-producing 
 t cells have been classified as a new 
 effector t - cell subset , termed th17 , 
 which is distinct from th1 , th2 , and treg 
 subsets . it is very interesting to 
 examine whether pde4 inhibitors 
 suppresses the il-23 or transforming growth 
 factor ( tgf- ) production from macrophages . il-23 , il-6 , and tgf- promote the differentiation 
 of th17 cells , 
 which are considered to 
 act as the key effector - cell 
 subset in inflammatory arthritis , 
 at least in rodents . in conclusion , this 
 study provides a unique murine model to 
 easily evaluate the effects of the drugs 
 on the formation of the granuloma and 
 cytokine production in a granuloma . pde4 inhibitors 
 showed an effect comparable to those of 
 antirheumatic drugs in this model . the 
 suppression of tnf- and il-12 production 
 and the inhibition of fibroblast 
 proliferation were considered to contribute 
 to the antipannus forming effects 
 of pde4 inhibitors .
a marked proliferation of synovial fibroblasts in joints leads to pannus formation in rheumatoid arthritis ( ra ) . 
 various kinds of cytokines are produced in the pannus . 
 the purpose of this study is to elucidate the effects of phosphodiesterase 4 ( pde4 ) inhibitors in a new animal model for the evaluation of pannus formation and cytokine production in the pannus . 
 mice sensitized with methylated bovine serum albumin ( mbsa ) were challenged by subcutaneous implantation of a membrane filter soaked in mbsa solution in the back of the mice . 
 drugs were orally administered for 10 days . 
 the granuloma formed around the filter was collected on day 11 . 
 it was chopped into pieces and cultured in vitro for 24 hr . 
 the cytokines were measured in the supernatants . 
 the type of cytokines produced in the granuloma was quite similar to those produced in pannus in ra . 
 both pde4 inhibitors , kf66490 and sb207499 , suppressed the production of il-1 , tnf- , and il-12 , and the increase in myeloperoxidase activity , a marker enzyme for neutrophils and hydroxyproline content . compared to leflunomide , pde4 inhibitors more strongly suppressed il-12 production and the increase in myeloperoxidase activity . 
 pde4 inhibitors also inhibited lipopolysaccharide - induced tnf- and il-12 production from thioglycolate - induced murine peritoneal macrophages and the proliferation of rat synovial fibroblasts . 
 these results indicate this model makes it easy to evaluate the effect of drugs on various cytokine productions in a granuloma without any purification step and may be a relevant model for evaluating novel antirheumatic drugs on pannus formation in ra . 
 pde4 inhibitors could have therapeutic effects on pannus formation in ra by inhibition of cytokine production by macrophages and synovial fibroblast proliferation .