graves ' orbitopathy ( go ) is a disfiguring and sometimes blinding disease , characterized by inflammation and swelling of orbital tissues , with fibrosis and adipogenesis being predominant features . clearly , the vulnerability to disease manifestation most likely reflects the highly specialized function of the orbital tissue , a unique fat depot that cushions the globe . found that , in go patients , 25% have orbital fat and muscle volumes within an age - specific reference range . an increase of the fat volume , characterized by proptosis , is seen in approximately 14% of go patients . however , only 3 to 5% of patients with go have severe disease with intense pain , inflammation , and sight - threatening corneal ulceration or compressive optic neuropathy . the sparse mononuclear cell infiltrates are seen within the fatty connective tissue and the muscle endomysium [ 5 , 6 ] . in the early inflammatory phase the majority of cells are t lymphocytes cd4 + and cd8 + and b lymphocytes are only occasionally seen [ 7 , 8 ] . macrophages influx is increased in the early disease and less so in late disease . the inflammatory cells , t and b lymphocytes , and macrophages as well as mast cells infiltrating the orbit interact with orbital fibroblasts and amplify inflammatory / autoimmune reaction . activated human t lymphocytes by expressing cyclooxygenase-2 and producing prostaglandins drive human orbital fibroblast differentiation to adipocytes . moreover , engagement of cd40 on orbital fibroblasts triggers hyaluronan synthesis and activation of inflammatory cyclooxygenases . when exposed to tgf- , thy-1 fibroblast differentiates into myofibroblasts with prominent cytoplasmic actin filaments that can participate in inflammation , repair , and fibrosis and are responsible for tissue remodeling in go [ 12 , 13 ] ( figure 1 ) . tgf- and other inflammatory mediators ( il-16 , rantes ) elaborated by resident macrophages and fibroblasts trigger t cells migration or participate directly in local inflammation . the inflammatory cyclooxygenase ( cox2 ) is usually expressed at extremely low levels under normal basal physiology conditions . cox2 is expressed at higher levels in the orbital fibroadipose tissue in go ; there seems to be a positive correlation with increasing severity of ophthalmopathy , suggesting a possible relationship with cox2 expression and orbital inflammation in go [ 15 , 16 ] . of the cell types residing in go tissue preadipocytes and fibroblasts are most likely target and effectors cells of the orbital immune processes . fibrogenic growth factor , oxygen free radicals , and cytokines released from inflammatory cells act upon orbital preadipocytes , in para- or autocrine manner , stimulating adipogenesis , fibroblast proliferation , and glycosaminoglycan synthesis . expression of fgf- in the orbital fat / connective tissue with go and a direct relation with higher cas suggest its important role in severity of go . therefore we decided to perform a study aimed at comparing leucocyte infiltration pattern and inflammatory ( cox2 ) and profibrotic ( fgf- , tgf- ) cytokine milieu in the orbital fat / connective tissue of patients with mild and severe go taken during decompression surgery . human orbital tissue was obtained from 27 patients with go ( 26 females and 1 male ) classified according to the european group on graves ' orbitopathy , who underwent orbital decompression procedures , from the orbital tissue bank at the department of ophthalmology and university of essen . the mean age of patients at the time of surgery was 44.5 years ( range 2647 ) . control fat / connective tissues were derived from 10 individuals ( 9 females and 1 male ) undergoing orbital surgery for blepharoplasty with no history of go or any orbital inflammatory disease . surgical specimens of orbital fat / connective tissue were obtained and immediately snap - frozen in liquid nitrogen until use . the clinical activity score of go ( cas ) was estimated according to mourits et al . . the severity of the eye disease was estimated using nospecs classification ( no signs or no symptoms ; only signs , no symptoms ; signs only ; proptosis ; eye muscle involvement ; corneal involvement ; and sight visual acuity reduction ) . vi ) required orbital bony decompression due to optic nerve compression and limited extraocular muscle functions ( 22 specimens ) . mean duration of thyroid disease was 2.5 ( sd 1.5 ) years with 1.2 ( sd 1.0 ) years for go . before surgery all patients had received > 1 cycles of steroid regiment , and all but 2 received orbital irradiation . patients with mild go ( n = 9 ) ( nospecs iii - iv ) underwent orbital bony decompression to reduce proptosis or for fat resection . mean duration of thyroid disease was 3.9 ( sd 2.0 ) years and 3.3 ( sd 1.9 ) for go . evaluation of proteins expression was done using immunohistochemical methods . following the deparaffinisation and rehydration , epitope retrieval was carried out in the envision flex target retrieval solution ( dako ) in high ph . endogenous peroxidases were blocked by incubating the sections in methanol and 3% hydrogen peroxidase for 20 minutes . visualization reagent envision flex ( dako ) was applied for 30 minutes followed by dab solution for 10 minutes . the results were expressed as the percentage of cells with a strong positive staining as follows : 10% positive cells  negative (  ) , between 11% and 50% ( + ) , and 51% positive cells ( + + ) . in mild type of go we observed t lymphocytes cd4 positive ( t helpers ) within the tissue in 6 out of 9 mild cases ; mostly t cells were dispersed within the whole tissue and next to the small blood vessels . in 2 specimens t helpers were together with t cytotoxic cells ( cd8 positive ) ; however t cd8 were in minority . in 3 t cells were mostly cd4 positive t helpers ; only in 2 cases we also observed few cd8 positive t cytotoxic cells . we did not observed presence of b lymphocytes in almost all specimens of both severe and mild go ( with only 1 exception of severe go , where we observed a focal infiltration of b lymphocytes ) . in all mild the cells were dispersed in whole tissue , especially in more fibrotic connective tissue , rather than in fat one . in 4 out of 9 cases the cells were numerous and prominent ( see table 2 ) . in all severe go specimens we observed presence of macrophages and fibroblasts ; mostly the cells were numerous and were disseminated in whole tissue ( in 15 cases we evaluated the staining as + + ) . number of patients and the percentage of cd68 expression within score 0 , 1 , 2 in examined groups ( see table 3 ) . the explanation of this may by associated with the previous steroid therapy in almost all go patients . in all mild go tissues we observed expression of fgf-. the staining was observed mainly in adipocytes and fibroblasts within the tissue . however only 2 out of 9 mild go were evaluated as ( + + ) . all of the severe go cases showed fgf- expression within the adipose tissue as well as in the fibroblasts in the connective tissue . also fgf- expression was observed in numerous small blood vessels . in almost all of the examined severe go cases ( 15 specimens ) fgf- expression was estimated as ( + + ) . number of patients and the percentage of fgf- expression within score 0 , 1 , 2 in examined groups ( see table 3 ) . all of the examined mild go specimens showed tgf- expression mainly within the fibroblasts of the connective fibrous tissue ; however the expression was mostly dispersed . most of the examined severe go specimens presented strong and diffused pattern of the tgf- expression , all in the numerous fibroblasts within the connective fibrous tissue . there was a positive correlation of cas values with cd4 and fgf- , tgf- , and cd68 expression ( see table 4 ) . retrobulbar tissue specimens are generally not available from patients with early , active go without prior immunomodulatory treatment ( with glucocorticoids or radiotherapy ) . they demonstrated that both cd4 + and cd8 + cells were present , and a significant proportion of them were cd45ro+ cells . infiltration of oct ( orbital connective tissue ) by hla - dr+ , cd25 + , and tnf- cells suggests that th1-type immune reaction with the interference of proinflammatory cytokine(s ) ( tnf- ) may be important in the pathogenesis of disease . recently an elevation of cd4 to cd8 ratio and enhanced secretion of il-6 , il-10 , and tnf were detected in pbmcs of go patients compared with controls . similar to our study , previously the examined orbital tissue specimens from patients with go who had received immunosuppressive therapy have shown predominant cd4 + infiltration and only 2030% of infiltrating cells were cd3+cd8 + cells [ 23 , 24 ] . examined orbital oct - derived t cell lines from go using immunohistochemical methods , and they reported that t cell marker cd3 + could be detected in almost 100% of cases . similar results were found by yang and coworkers who established 104 t cell clones from oct biopsies of 3 patients with go and found that approximately 7080% were cd3+cd4 + and approximately 2030% were cd3+cd8 + cells . nevertheless , in both published papers all examined patients had been treated either with systemic steroids and/or orbital radiotherapy . in the previous study , eckstein et al . have shown the predominant intraorbital cd4 + t cells infiltration in go with absence of cd8 + and cd20 + b lymphocytes . chen et al . found that macrophage infiltration may play an important role in the pathogenesis of go via overexpression of mcp-1 . they also found that the expression of mcp-1 was higher in go orbital fat than in the orbital fat of controls . in our study we demonstrated cd68 macrophages ' infiltration in fibrous connective and fat tissue more expressed in severe than mild go ( figures 3(c ) and 3(d ) ) . previously , eckstein et al . have documented in the orbital tissue an increase of immigrant macrophages cd14 and rfd7 influx . the presence of such cells in go with strong correlation to disease activity suggests robust proinflammatory secretion on site . concluded that these macrophages are recruited from freshly infiltrated monocytes , but not local resident macrophages . recently it has been found that , in go , mast cells , monocytes , and macrophages may activate orbital fibroblasts via secretion of especially pdgf - ab and pdgf - bb . matos et al . have investigated the immunohistochemical expression of growth factors ( igf-1 , pdgf - a , pdgf - b , fgf , and vegf ) in patients with graves ' ophthalmopathy . there was a direct relation with higher cas ( clinical activity score ) in all of them . when cas equal or higher than 5 was considered , the percentage of igf-1 expression was 54.5% . fgf expression was in 5 cases ( 20.8% ) with a direct relation in all those with higher cas ( > 5 ) , suggesting its important role in active go ( 45.4% ) . they concluded that in all patients , except one , with positive expression of fgf , igf-1 and vegf showed cas greater than 5 , suggesting in this way an important role of these growth factors in the pathogenesis and severity of graves ' ophthalmopathy . significant proliferation of the fibrous connective tissue in severe go was seen in our specimens ( figure 2(c ) ) . in addition to our study an increased expression of fgf in the fibroblasts ( figure 4(a ) ) and adipocytes ( figure 4(b ) ) was observed in the connective tissue of the severe go . yi and xu observed high cox2 expression in thyroid - associated ophthalmopathy and bloise et al . proved treatment of moderate go with oral sodium diclofenac is a good and safe therapeutic option [ 28 , 29 ] . moreover , they found that diclofenac , an inhibitor of cyclooxygenases with antagonistic effects on ppar - gamma , reduced the number of mature adipocytes by approximately 50% . recently attenuation of interleukin- ( il- ) 1-induced cyclooxygenase- ( cox- ) 2 and prostaglandin ( pg)e2 expression in orbital fibroblasts from patients with thyroid - associated ophthalmopathy ( tao ) has been proven to decrease inflammation . this statement would be in agreement with our results , showing no cox-2 expression in our specimens , since most of our studied patients received prior to orbital decompression glucocorticosteroids that may had hampered the inflammation mandated by cox2 . macrophages and cd4 t lymphocytes are both engaged in the active / severe and long stage of inflammation in the orbital tissue . fgf- and tgf- expression may contribute to tissue remodeling , fibrosis , and perpetuation of inflammation in the orbital tissue of go especially in severe go .
purpose . to assess fgf- , tgf- , and cox2 expression and immunocompetent cells in the orbital tissue of patients with severe and mild graves ' orbitopathy . 
 patients and methods . 
 orbital tissue was taken from 27 patients with go : ( 1 ) severe go ( n = 18 ) , the mean clinical activity score ( cas ) being 8.5 ( sd 2.5 ) ; and ( 2 ) mild go ( n = 9 ) , the mean cas being 2.2 ( sd 0.8 ) , and from 10 individuals undergoing blepharoplasty . 
 the expression of cd4 + , cd8 + , cd20 + , and cd68 and fgf- , tgf- , and cox2 in the orbital tissue was evaluated by immunohistochemical methods . 
 results . 
 we demonstrated predominant cd4 + t cells in severe go . 
 cd68 expression was observed in the fibrous connective area of mild go and was robust in severe go , while the prominent tgf- expression was seen in all go . 
 increased fgf- expression was observed in the fibroblasts and adipocytes of severe go . 
 no expression of cox2 was found in patients with go . 
 conclusions . 
 macrophages and cd4 t lymphocytes are both engaged in the active / severe and long stage of inflammation in the orbital tissue . fgf- and tgf- expression may contribute to tissue remodeling , fibrosis , and perpetuation of inflammation in the orbital tissue of go especially in severe go .