systemic lupus erythematosus ( sle ) is a multisystem , autoimmune , connective - tissue disorder in which organs and cells undergo damage mediated by tissue - binding autoantibodies and immune complexes . t cells from sle patients are known to provide help to b cells to produce autoantibodies and numerous abnormalities of t cells including aberrancies of proliferation , cell death , signaling , and cytokines production have been described in the pathogenesis of sle [ 2 , 3 ] . lipid rafts are liquid ordered sphingolipid and cholesterol - enriched membrane domains , functioning in cellular processes , especially in signal transduction through recruiting signaling and stimulatory proteins , and play a critical role in t lymphocytes activation , particularly in signaling from the t - cell antigen receptor ( tcr ) and in localization and function of proteins residing proximal to the receptor     [ 47 ] . ganglioside gm1 , a major constituent of cellular membranes acting as a rigid barrier to the extracellular environment , is one of the important component of lipid raft . high levels of gm1 and cholesterol have been found in peripheral blood t cells from sle patients , which was only measured in whole negatively selected t cells population by confocal microscopy study . however , the levels of gm1 in t cell subgroups such as cd4 + helper t cells and cd8 + cytotoxic t cells are largely unknown . cd30 , a 120-kda type i transmembrane glycoprotein , is normally found on a subset of activated t cells , which are involved in the induction of cell proliferation and initiation of apoptosis    . the soluble form of cd30 ( scd30 ) is released from activated t cells through proteolytic cleavage . elevated serum levels of scd30 have been found in hodgkin 's disease , anaplastic large cell lymphoma , infectious and allergic diseases as well as some autoimmune diseases , such as sle and rheumatoid arthritis     [ 11 , 12 ] . however , its relationship with the membrane raft gm1 and cytokines in sle has not been defined . to analyze lipid raft expression on each subgroup t cells in sle and its relation to abnormal t cell activation and cytokine production , we quantified gm1 expression on both peripheral cd4 + and cd8 + t cells from the sle patients via flow cytometry and compared it to the serum levels of scd30 and th1/th2 balance of cytokines as well as clinical parameters . we found that gm1 expression was increased on cd4 + t cells and there were significant correlations between gm1 expression and scd30 and disease activity in sle . forty - four consecutive patients fulfilling the revised american college of rheumatology ( acr ) criteria for the diagnosis of sle   were recruited for this study . disease activity was scored and the sle disease activity index ( sledai ) was calculated based on previous report . patients were divided into subgroups according to clinical disease activity ( i.e. , active 10 and inactive < 10 by sledai ) and serum igg level ( high igg > 15 our study included 21 active sle patients , 23 inactive patients with sledai ranging from one to 20 , and 28 healthy control volunteers . complete blood cell count , serum complement , serum igg , antinuclear and anti - dna antibodies were measured in all patients . surface expression of cd4 , cd8 , cd45ro , and gm1 were analyzed from the peripheral blood cells by double or triple - staining using fitc- , pe- , apc - labeled mabs in the relevant combinations . two color immunofluorescence analysis was performed on a facscan flow cytometer ( bd biosciences , san jose , ca ) , using cellquest pro ( bd biosciences ) software . detection of gm1 expression was performed by staining peripheral blood with pe - conjugated anti - cd4 or cd8 antibodies ( ebioscience co. ltd , usa ) and fitc - conjugated cholera toxin - b ( ctb , sigma - aldrich , usa ) as described previously . for triple staining , cells were first analyzed by apc - conjugated cd4 , then gm1 expression was detected with pe anti - cd45ro antibody and fitc cholera toxin - b . mononuclear cells were isolated from heparinized peripheral blood from the healthy subjects by density - gradient centrifugation on ficoll - hypaque ( pharmacia , uppsala , sweden ) . the cells were then stimulated with 1  g / ml of pha ( boehringer mannheim , germany ) and maintained in rpmi 1640 medium supplemented with 10% fcs ( upstate biotechnology , inc . , ubi , lake placid , ny ) , 2  mm l - glutamine , penicillin , and streptomycin in the presence of 2  nm recombinant il-2 ( shionogi co. ltd . , osaka , japan ) for 2 weeks . to analyze surface expression of ganglioside gm1 and cd45ro on cd4 + t cells , cells were stained with fitc - conjugated ctb ( sigma - aldrich ) , pe - conjugated anti - cd45ro and apc - conjugated anti - cd4 mabs ( bd bioscience ) on ice for 30 minutes , respectively , and analyzed with a facsan . the serum levels of scd30 , il-10 , and tnf- were measured by an enzyme - linked immunosorbent assay ( elisa ) commercial kit ( r&d co. ltd . the serum igg of the sle patients was detected by immunoturbidimetric assay ( orion diagnostica oy co. ltd . the results were subjected to the independent sample t - test , correlation tests , and bivariate analysis . a value of p < .05 was considered to be statistically significant . ctb binds the raft - associated gm1 , previously shown to be a reliable marker for detection of lipid raft domains . first , we analyzed the rafts on mitogen - stimulated t cells in vitro through facs . gm1 surface expression with fluorescent - labeled ctb was determined on days 0 , 2 , 5 , 8 , 11 and 14 after stimulation with 1  g / ml of pha . the mean fluorescence intensity ( mfi ) of memory t cells ( cd45ro / cd4 ) were dramatically increased in time - dependent manner after stimulation ( figures 1(a ) and 1(b ) ) as well as cd4 + t cells ( figure 1(c ) ) . these data indicate that cellular membrane adopts an increased lipid raft structure upon mitogenic stimulation in cd4 + t cells and cd4 + memory t cells . next , we examined the expression of gm1 on peripheral blood cd4 + t cells and cd8 + t cells from 44 patients with sle and 28 healthy controls by flow cytometry . the mfi of gm1 on peripheral cd4 + t cells from patients with sle were significantly stronger than those from the healthy controls ( p < .01 ) ( figure 2(a ) ) . although the increase of cd4 + t cells expressing gm1 was small , the substantial change in gm1 expression as measured by mfi suggests that increased gm1 expression affects a high proportion of lupus cd4 + t cells . when we divided sle patients into active group ( n = 21 ) and inactive group ( n = 23 ) according to sledai scores ( active 10 , inactive < 10 ) , mfi of gm1 in both groups are higher than those of healthy controls . although the difference was not significant , mfi of gm1 in active group is higher than that in inactive group ( figure 2(a ) ) . when we divided sle patients into high igg group ( n = 24 ) and normal igg group ( n = 20 ) according to serum igg level ( > 15 g / l ) , mfi of gm1 on cd4 + t cells from high igg group was statistically higher than that from normal igg group ( p < .05 ) ( figure 2(a ) ) . in contrast to cd4 + t cells , no significant difference in expression of gm1 on cd8 + t cells was observed between sle patients and the healthy controls ( figure 2(b ) ) . further , we analyzed gm1 expression on cd45ro+/cd4 + memory t cells from 11 of inactive sle , 14 of active sle patients and 23 healthy controls by triple staining ( figures 2(c)2(e ) ) . figure 2(c ) shows that there are no significant differences in percentage of cd4 t cells among inactive sle ( 42.2  3.04 ) , active sle ( 38.8  2.64 ) , and healthy controls ( 46.9  2.17 ) . we did not found the significant difference of the mfi of gm1 on cd45ro+/cd4 + memory t cells among each group ( data not shown ) . however , when we determined the cell with more than 10 in mfi of gm1 as high gm1 cells ( figure 2(d ) ) , percent of high gm1 cells among cd45ro+/cd4 + memory t cells was significantly increased in sle patients ( 1.00  0.07 and 1.35  0.12 for inactive and active sle , resp . ) compared with healthy controls ( 0.69  0.03 ) ( figure 2(e ) ) . although the change in the percentage of high gm1 cells was small , this represented a twofold increase compared with those from healthy individuals . moreover , percent of high gm1 cells in active sle is significantly higher than that in inactive sle ( figure 2(e ) ) . the elevation in high gm1 cells of memory t cells was dependent of lupus disease activity status . the serum levels of scd30 were averaged 149.74  63.85  ng / ml in the sle patients ( 149.25  66.83 ng / ml in active patients , 150.3  62.65  ng / ml in inactive patients ) , and were averaged 79.49  22.93  ng / ml in the healthy controls , showing a significant increase of serum scd30 in the sle patients , compared to healthy controls ( p < .01 ) , although there was not difference between sle active and inactive groups ( figure 3(a ) ) . levels of il-10 were 94.6  57.93  pg / ml in the sle patients ( 89.07  46.44  pg / ml in active patients , 100.86  69.9 pg / ml in inactive patients ) and 44.96  33.30  pg / ml in the healthy controls . there were statistical differences between the sle patients and healthy controls ( p < .05 ) ( figure 3(b ) ) . tnf- were 18.62  9.57  pg / ml in the sle patients ( 19.35  9.15  pg / ml in the active patients , 17.80  10.20  pg / ml in the inactive patients ) and 16.91  13.18  pg / ml in the healthy controls . there were no statistical differences in the levels of tnf- between the sle patients and the healthy controls ( figure 3(c ) ) . analyzing the correlations among the sle patients , we found that there was a significant correlation between gm1 expression on cd4 + t cells ( mfi ) , sledai scores(r = 0.556 , p < .005 ) , plasma level of scd30 ( r = 0.51 , p < .005 ) , and serum igg levels ( r = 0.56 , p < .005 ) in the sle patients . in particular , the correlation between mfi of gm1 on cd4 + t cells and sledai was much stronger in active sle group ( r = 0.813 , p < .001 ) than those in inactive group ( r = 0.668 , p < .005 ) . no significant correlation was found between gm1 and il-10 , ana titer , c3 , and other clinic parameters ( data not shown ) . furthermore , we found that the plasma level of scd30 was positively correlated with the igg level ( table 1 ) . but no statistical difference was observed between scd30 levels and other parameters ( data not shown ) . in addition , no statistical difference was observed between expression of gm1 on cd45ro+/cd4 + memory t cells and above parameters in patients with sle . t - cell abnormalities and aberrant t helper cytokine profiles have been implicated in pathogenic process of sle . previous studies have demonstrated that negative selected peripheral blood t cells from the sle patients have higher levels of gm1 . in this study , we quantitatively measured membrane gm1 on peripheral blood cd4 + and cd8 + t cells , respectively , and found that cd4 + t cells expressed elevated gm1 , but cd8 + t cells did not , in sle patients . in addition , gm1 expressions on memory t cells ( cd45ro / cd4 ) and cd4 + t cells were dramaticly increased after pha stimulation in vitro . furthermore , number of gm1-highly expressing cd45ro+cd4 + memory t cells were significantly increased in sle patients compared with healthy controls ( figure 2(e ) ) . these data may indicate that cellular membrane adopts an increased lipid raft structure upon mitogenic stimulation and cd4 + t cells in sle patients are continuously activated by autoantigens in vivo . it has been reported that cd8 + t cells in sle are impaired and   cd8 + t cells do not require the polarization of lipid rafts for activation and proliferation    . however , activated cd4 + helper t cells produce more cytokines and help b cells secrete autoantibodies that form immune complexes , resulting in subsequent inflammation and organ damage . additionally , we found that there was a positive correlation between gm1 expression on cd4 + t cells ( mfi ) and sledai scores ( r = 0.556 , p < .005 ) as well as serum igg levels ( r = 0.56 , p < .005 ) in sle group , suggesting a possible role of lipid raft gm1 in pathogenesis of sle . it has been documented that sle t cells possess dysregulated activity of multiple protein tyrosine kinases     [ 9 , 20 , 21 ] . we hypothesize that high level of gm1 on cd4 + t cells may induce changes in membrane density and segmentation impacting on signal strength . previously , we reported that lipid rafts function as scaffolds for tcr signals by recruiting tcr , zap-70 and pkc- into rafts using membrane sphingomyelin knock down jurkat cells . these modulations may also facilitate that signaling , and the stimulatory proteins are recruited to or modified in lipid rafts , subsequently convey aberrant intracellular signals , resulting in abnormal cd4 + t cell function . these aberrances lead to the survival of autoimmune b clones and overproduction of antibodies , eventually contribute to the development of sle . although cd30 originally described as a marker on reed - sternberg cells in hodgkin lymphoma     , cd30 can be found on various populations of t cells ( both cd4 + and cd8 + cells ) , especially helper t cells which produce cytokines . the extracellular portion of cd30 is proteolytically cleaved from cd30 + cells , possibly on activation by cd30l , to produce a soluble form of the molecule ( scd30 ) detectable in serum . the scd30 belongs to the tnf superfamily and is important in the interaction and the activation of t and b cells . high levels of scd30 have been seen in the cases of t helper immunodeficiency diseases , allergic diseases , the severe infections and autoimmune disease , and scd30 has been proposed as a marker for a th2-oriented immune response     [ 12 , 24 ] . reported that there was a significant differences of scd30 at serum levels between the sle patients and the controls . we also found significant differences of serum scd30 level between the sle patients and healthy controls . moreover , we found that there was a positive correlation between gm1 expression on cd4 + t cells ( mfi ) and serum level of scd30 ( r = 0.51 , p < .005 ) . a triton x-100-based method for lipid raft isolation revealed that cd30 was partially present in lipid rafts     .additionally , bastian von tresckow et al . found that cellular lipid raft such as cholesterol level regulate cd30 shedding in human hodgkin lymphoma- and non - hodgkin lymphoma derived cell line     , implying the potential relationship between lipid raft and scd30 . recently , scd30 has been shown to be important for the regulation of the immune system in vivo and in vitro , and it is likely to play a regulatory role in the th1/th2 type immune response [ 26 , 27 ] . increased serum levels of scd30 in autoimmune disease demonstrated that the aberrant lipid raft is connected to an increased secretion of scd30 . we further measured the tnf- and il-10 which respectively indicate th1 and th2 activation     [ 2 , 3 , 26 ] . we found that the levels of il-10 were significantly higher in sle than those in healthy controls , however no significant difference of tnf- was observed . although the role of il-10 in sle pathogenesis has not been completely elucidated , il-10 is considered a regulatory th2 cytokine , and it causes activation , differentiation , and antibody production in b - cells . in addition , current studies reveal the existence of a subset of b cells with regulatory capacities . this regulatory subset is characterized by the secretion of il-10 and transforming growth factor- ( tgf- ) and has been shown to be involved in the pathogenesis of autoimmune diseases . however , we did not found the positive correlation between il-10 and scd30 , as well as other parameters . our data suggest that sle il-10 is complex and has pleiotropic effects in immunoregulation and inflammation .        taken together , we have found an aberrant gm1 expression on cd4 + t cells and significant correlations between gm1 expression and scd30 and disease activity in sle , indicating that lipid raft may become a direct target for modulation of the immune system .
gangliosides gm1 is a good marker of membrane microdomains ( lipid rafts ) with important function in cellular activation processes . in this study we found that gm1 expression on cd4 + t cells and memory t cells ( cd45ro / cd4 ) 
 were dramatic increased after stimulation with phytohaemagglutinin in vitro . 
 next , we examined the gm1 expression on peripheral blood cd4 + t cells and cd8 + t cells from 44 patients with sle and 28 healthy controls by flow cytometry . 
 gm1 expression was further analyzed with serum soluble cd30 ( scd30 ) , il-10 , tnf - alpha and clinical parameters . 
 the mean fluorescence intensity of gm1 on cd4 + t cells from patients with sle was significantly higher than those from healthy controls , but not on cd8 + t cells . 
 increased expression of gm1 was more marked on cd4+/cd45ro+ memory t cells from active sle patients . 
 patients with sle showed significantly elevated serum scd30 and il-10 , but not tnf - alpha levels . 
 in addition , we found that enhanced gm1 expression on cd4 + t cells from patients with sle positively correlated with high serum levels of scd30 and igg as well as disease activity ( sledai scores ) . 
 our data suggested the potential role of aberrant lipid raft / gm1 on cd4 + t cells and scd30 in the pathogenesis of sle .