hemolytic uremic syndrome ( hus ) is the most frequent cause of acute renal failure in children . the traditional diagnostic criteria for this syndrome include hemolytic anemia with fragmented erythrocytes , thrombocytopenia , and renal failure . the epidemic form of hus occurs mostly as a result of hemorrhagic colitis . in 90% of hus cases , infection with shiga toxin- ( stx- ) producing escherichia coli ( stec ) is strongly implicated . stx is divided into two main groups , referred to as stx1 and stx2 , which share approximately 56% amino acid sequence similarity . stx2 is a more potent cause of severe hus and central nervous system ( cns ) impairment . stx is a 71-kda protein composed of a 32-kda a subunit ( stxa ) and five 7.7-kda b subunits ( stxb ) . stxa has n - glycosidase activity that removes the adenine of the 28s rna within the 60s ribosomal subunit , thereby rendering ribosomes inactive for protein synthesis . stxb specifically binds to the cell membrane of the globotriaosylceramide ( gb3 ) receptor , which is present in specific mammalian host cells . stx is thought to enter the bloodstream through inflamed intestinal mucosa , then bind with low affinity to blood cells , specifically polymorphonuclear leukocytes , thereby being transported to the target organs . gb3 , the functional receptor for stx , is present on the surfaces of various cell types , including human glomerular endothelial cells , glomerular epithelial cells , and human brain endothelial cells ( hbecs ) . stx binding to target cells expressing gb3 inhibits protein synthesis and induces apoptosis and necrosis . approximately , 30% of patients with hus suffer from cns complications , and these patients have the poorest prognosis . although thrombotic microangiopathic damage is characteristic of hus and is thought to result from the direct cytotoxic effects of circulating stx on the vascular endothelium , there is no consensus on the pathogenic mechanisms responsible for brain involvement in hus . however , a possible role for the inflammatory response provoked by cytokines has been suggested . clinical studies have demonstrated that patients with hus have elevated plasma levels of cytokines such as tumor necrosis factor  ( tnf- ) , interleukin-1 ( il-1 ) , and interleukin-6 ( il-6 ) . in the presence of tnf- or il-1 , furthermore , tnf- and il-1 upregulate the expression of gb3 in endothelial cells . similarly , although it has been demonstrated that hbecs are very resistant to stx - induced cytotoxicity , inflammatory mediators , such as tnf- and/or il-1 , markedly increase the sensitivity of hbecs to stx cytotoxicity , which is due to modulation of expression of gb3 . several components of the gb3 metabolic pathway may be involved in cytokine - stimulated gb3 expression . gb3 is synthesized from lactosylceramide and udp - galactose by gb3 synthase ( galt6 ) . the inflammation associated with hus is marked by the release of chemokines , and the levels of il-8 and mcp-1 are significantly increased in urine samples collected from hus patients . purified stxs directly induce the expression of the neutrophil chemoattractant il-8 in human intestinal epithelial cells . these stx - induced increases in il-8 synthesis by intestinal epithelial cells could be important in augmenting the host mucosal inflammatory responses to stec infection . endothelial cells exposed to stx2 release inflammatory chemokines , such as il-8 and mcp-1 , which stimulate adhesion and transmigration of leukocytes . however , in brain parenchymal cells and neuroglial cells , expression of chemokines in response to stx has not been studied . intravenous inoculation of rabbits with stx2 caused severe cns injury associated with the invasion of stx2 through the bbb . some authors have reported that intracerebroventricular administration of stx2 causes neuronal death and glial cell damage in the rat brain . transmission electron microscopy studies have revealed apoptotic neurons , ultrastructural alterations of glia , and demyelinated fibers . these findings support the contention that stx2 has a direct effect on brain neuroglial cells . the brain contains neuronal , glial ( e.g. , astrocytes , microglia , and oligodendrocytes ) , and endothelial cells . human astrocytes ( hasts ) are the most abundant type of glial cell in the human brain . they play key roles in development , homeostasis , inflammatory responses , and repair of the cns by producing a wide variety of cytokines , chemokines , and growth factors and by expressing receptors for these molecules . these chemokines play important roles in development , growth , cell migration , production of free radicals , apoptosis , t - cell activation , neoplasia , inflammatory regulation in response to injury , wound healing , tissue repair , and macrophage recruitment , as well as in interactions with pathogens , including viruses [ 2629 ] . chemokines and their receptors play important roles in several neurodegenerative and inflammatory diseases of the cns , including trauma , stroke , alzheimer 's disease , multiple sclerosis , cns tumors , and human immunodeficiency virus encephalitis [ 3032 ] . we speculated that stxs would act directly on astrocytes by inducing the production of chemokines and play a role in cns complications .   in the present study , we investigated how gb3 expression , including galt6 , in hasts is controlled by inflammatory cytokines . in addition , we sought to obtain further evidence that stx2 acts to produce chemokines in hasts and that these chemokines participate in the pathogenesis of cns complications associated with hus . toxicity was determined to be greater than 10 units per milligram using a vero cell assay . all reagents were purchased from sigma chemical co. ( st . louis , mo , usa ) . hasts derived from human fetal brain tissue ( lonza , walkersville , md , usa ) were routinely subcultured every 6 days in clonetics astrocyte growth medium ( lonza ) . cells were reseeded after reaching confluence at 3,500 cells per square centimeter and were incubated at 37c in a humidified atmosphere containing 5% co2 . analysis of gb3 expression stimulated by il-1 was conducted by immunohistochemistry using the vectastain elite abc kit ( vector labs , burlingame , ca , usa ) . the cells were stimulated for 48  h with il-1 and fixed in 4% paraformaldehyde for 10  min at room temperature . slides were prepared and incubated in rat anti - human cd77 igm antibody ( 1  :  100 dilution ; beckman coulter , brea , ca , usa ) or pbs for 8  h at room temperature , then washed and incubated with a 1  :  100 dilution of secondary antibody ( supplied in the vectastain kit ) at room temperature for 1  h. the colorimetric reaction was obtained using 0.5  mg / ml diaminobenzidine ( dab ) substrate solution ( vector labs ) with 0.02% h2o2 . hasts grown in six - well plates were incubated in il-1 ( 10 to 10  mol / l ) or tnf- ( 10 to 10 mol / l ) . at 0 , 8 , and 24  h postinoculation , total rna was isolated using the rneasy mini kit ( qiagen , valencia , ca , usa ) , with an rnase - free dnase ( qiagen ) treatment for 30  min in accordance with the manufacturer 's instructions . rna was reverse transcribed to produce cdna using the prime script rt reagent kit ( takara bio , otsu , japan ) . real - time quantitative polymerase chain reaction ( pcr ) was performed using sybr green pcr master mix and the thermal cycler dice real - time system ( takara bio ) . the real - time pcr primers specific for galt-6 and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) are listed in table 1 . the pcr conditions were 95c for 10  s , followed by 40 cycles of 95c for 5  s and 60c for 30  s. the relative amounts of mrna were determined from the threshold cycle values of the reference gene , gapdh . all further calculations and statistical analyses were carried out based on these values and are referred to as relative expression ratios . hasts grown in six - well plates were incubated in 10 and 10  mol / l stx2 . at 0 , 24 , and 48  h postinoculation , cell supernatants from treated hasts the elisas for il-8 and monocyte chemoattractant protein-1 ( mcp-1 ) were performed using the corresponding quantikine colorimetric sandwich elisa kits ( r&d systems , minneapolis , mn , usa ) in accordance with the manufacturer 's protocol . chemokines were quantified by measurement of absorbance at 450  nm ( a450 ) with a 680 microtiter plate reader ( bio - rad laboratories , hercules , ca , usa ) . the assay sensitivities were 4  pg / ml ( il-8 ) and 5  pg / ml ( mcp-1 ) . hasts grown in six - well plates were incubated with either 10 or 10  mol / l stx2 , 10  mol / l stx2 with 10  mol / l il-1 , or 10  mol / l il-1 alone . at 0 , 8 , and 24  h postinoculation , total rna was extracted and real - time quantitative pcr performed using the methods described previously . the real - time pcr primers specific for il-8 and mcp-1 ( takara bio ) are listed in table 1 . the pcr conditions were one cycle at 95c for 10  s , followed by 40 cycles of 95c for 5  s and 60c for 30  s. the relative amounts of the mrna were determined as described previously . the means  sd were determined , and the mean values in the two separate groups were compared using a student 's t - test . proinflammatory cytokines increase the expression of stx receptor gb3 on the surface of different stx target cells . to determine the levels of gb3 expression in hasts treated with il-1 treated for 48  h with 10  mol / l il-1 , a relatively high level of gb3 ( figure 1(a ) ) was detected in comparison to the untreated controls ( figure 1(b ) ) . conversely , no change in gb3 level was detected in hasts treated with tnf- ( data were not shown ) . it has been demonstrated using hbecs and intestinal epithelial cells that inflammatory cytokines increase gb3 expression levels by generating lactosylceramide through the activation of galt6 . immunohistochemistry revealed that the expression of gb3 in hasts was increased by il-1. in addition , we investigated the expression of galt6 mrna relative to that of the untreated controls following treatment with il-1 ( 10 to 10  mol / l ) or tnf- ( 10 to 10  mol / l ) for 8 or 24  h. at 8  h , il-1 ( 10 to 10  mol / l ) treatment resulted in significant induction ( p < 0.05 ) of galt6 mrna relative to the untreated controls ( figure 2(a ) ) . a 6.5-fold increase in the level of galt6 mrna was induced with 10  mol / l il-1. tnf- treatment showed a similar trend to il-1 treatment at 8  h , but it was not significant ( figure 2(b ) ) . astrocytes are an important source of chemokines that play a role in brain inflammatory responses . to investigate whether expression of il-8 and mcp-1 mrna was increased in hasts treated with stx2 , we used real - time pcr . following treatment with 10 to 10  mol / l stx2 for 8 or 24  h , real - time quantitative pcr was performed to verify the expression of il-8 and mcp-1 transcripts . following treatment with stx2 , il-8 mrna induction was significantly elevated ( p < 0.05 ) relative to the untreated controls ( figure 3(a ) ) . a 256-fold increase in the level of il-8 mrna was induced by 10  mol / l stx2 , and a 1,155-fold increase was induced with 10  mol / l stx2 at 8  h. a 5.7-fold increase of mcp-1 mrna was induced by 10  mol / l stx2 , and an 11.6-fold increase was induced with 10  mol / l stx2 at 8  h , in comparison with the untreated controls ( figure 3(b ) ) .   to determine whether the upregulation of chemokine mrna was accompanied by release of these chemokines into the medium , hasts were exposed to 10 mol / l and 10  mol / l stx2 for 24 and 48  h , and the concentrations of chemokines in the medium were measured by elisa . treatment of hasts with 10  mol / l and 10  mol / l stx2 increased secretion of il-8 into the medium relative to the control cultures at 24 and 48  h ( figure 4(a ) ) . the concentrations of il-8 in the medium were significantly higher in the stx2-treated cultures , especially for 10  mol / l stx2 , than those in the control cultures at 24 and 48  h. similarly , treatment of hasts with 10  mol / l and 10  mol / l stx2 increased the secretion of mcp-1 into the medium relative to the control cultures at 24 and 48  h ( figure 4(b ) ) . unlike il-8 , mcp-1 was released in the absence of stx2 stimulation ( figure 4(b ) ) .   using real - time pcr , we found that the expression of galt6 mrna was increased by stimulation with il-1 , and immunohistochemistry demonstrated that the level of gb3 expression in hasts was also increased by il-1 stimulation . to investigate whether the increase of gb3 expression induced by il-1 stimulation correlated with the expression of chemokine mrna , we treated hasts with stx2 and il-1. induction of il-8 mrna upon stimulation with 10  mol / l stx2 and 10  mol / l il-1 combined was significantly greater ( p < 0.05 ) at 24  h than that resulting from stimulation with either agent alone at the same concentration ( figure 5 ) . a 3,700-fold increase was induced by this combination of stx2 and il-1 , in comparison with the control levels . coincubation of stx2 with il-1 did not elicit a significant increase in the level of mcp-1 mrna ( data were not shown ) . although the pathogenesis of cns involvement in hus is not fully understood , the disruption and increased permeability of the bbb and neuronal disturbance resulting from hbec injury are central events in cns complications observed during the acute phase of hus . in a previous histopathologic study , mizuguchi et al . showed that intravenous administration of stx2 to rabbits resulted in hemorrhage and necrosis of both neural and endothelial cells in the brain . moreover , an in vivo study has shown that stx2 injected intravenously into rabbits was detected in the cerebrospinal fluid ( csf ) and that influx of stx2 into the csf caused injury to the tight junctions between ependymal cells , resulting in destruction of the bbb . some authors have reported that intracerebroventricular administration of stx2 causes neuronal death and glial cell damage in the rat brain . however , it is unclear how stx2 affects neuroglial cells during an inflammatory response . hasts comprise 55%60% of the total number of human brain cells   and are involved in virtually every type of brain pathology . hasts play key roles in the development , inflammation , and repair of the cns by producing a wide variety of chemokines . under inflammatory or otherwise pathologic conditions , hasts become activated and display enhanced production of several cytokines , chemokines , and growth factors . in addition , hasts generally start to proliferate under such conditions , a phenomenon known as astrogliosis . therefore , knowledge of the cellular response of hasts to stx is essential to understand the neuropathology observed in the severe cases of hus . in this study , il-1 treatment of hasts increased both gb3 and galt6 mrna expression , as determined by immunohistochemistry and real - time pcr , respectively . in addition , treatment of hasts with stx2 induced production of the chemokines il-8 and mcp-1 . these findings suggest that stx2 triggers an immune response through the expression of chemokines in hasts . toxin binding to gb3 is the primary determinant of the cytotoxic and pathologic effects of stx protein . previous studies have highlighted the role of proinflammatory agents in enhancing the sensitivity of different target cells , including hbecs , to the effects of stx through upregulation of gb3 expression [ 9 , 35 ] . primary hbecs become susceptible to stx when they express gb3 in response to tnf- , further exacerbating the disease process . therefore , we investigated whether gb3 expression in hasts is increased by inflammatory cytokines . using real - time pcr , we determined that the expression of galt6 mrna was increased by il-1 stimulation , and immunohistochemistry showed that the level of gb3 expression in hasts was increased by il-1 stimulation . these results demonstrated that hasts , which are cns parenchymal cells , express gb3 , and that this expression is increased by inflammatory cytokines . in hbecs , il-1 and tnf- increased the expression of gb3 and enhanced the toxicity of stx . only il-1 increased the expression of galt6 mrna and increased the level of gb3 expression in hasts . treatment with tnf- elicited a response similar to that for il-1 in terms of the expression of galt6 mrna at 8  h , although the degree of similarity was not significant . in this study , the mechanism by which il-1 increased the level of gb3 expression in hasts more than tnf- is not understood , although it may be a result of differences in cell types , and further investigations of this are required . chemokines , a family of proinflammatory cytokines , are divided into two subfamilies : the cxc family , represented in this study by il-8 , and the cc family , represented here by mcp-1 . chemokines stimulate target cell - specific directional migration of leukocytes and may be potent mediators of inflammatory processes . human il-8 is a proinflammatory chemokine secreted predominantly by monocytes , endothelial cells , and glial cells and acts as a chemoattractant and activator of neutrophils via cxcr1 and cxcr2 receptor signaling . mcp-1 is a potent chemotactic factor for monocytes and plays an important role in the regulation of repair processes and cellular interactions in the cns [ 25 , 26 ] . it can be expressed by many cell types including microglia , astrocytes , and neurons [ 20 , 21 ] . in recent years , it has been shown that chemokines play many roles in inflammation in the cns and are responsible for the development of various neurodegenerative diseases and ischemic damage to brain cells [ 17 , 18 ] . in addition , the levels of mcp-1 and il-8 are significantly elevated in the csf of infants with amyotrophic lateral sclerosis , japanese encephalitis , or hypoxia - ischemia . we found that by stimulating hasts with stx2 chemokines such as il-8 and mcp-1 were produced , the production of il-8 being particularly marked . elevated levels of il-8 and mcp-1 mrna were found in toxin - treated cells in comparison to the untreated controls . il-8 and mcp-1 proteins were found at higher levels in the conditioned media of stx2-treated cells than in those of untreated cells , and the chemokines il-8 and mcp-1 were produced by hasts upon stimulation with stx . several studies have demonstrated that stx induces the expression of il-8 and mcp-1 in intestinal epithelial cells , endothelial cells , and monocytes and that the levels of il-8 and mcp-1 are significantly increased in urine samples collected from patients with hus . it has , therefore , been suggested that chemokines such as il-8 and mcp-1 play a specific role in the pathogenesis of hus . for hus patients , a high peripheral polymorphonuclear neutrophil count at presentation activated polymorphonuclear neutrophils could contribute to the brain inflammatory response and participate in the secondary damage following bbb disruption in hus . stx2 induces expression of several chemokines , such as il-8 and mcp-1 , in astrocytes , and might constitutively induce the inflammatory response . in a mouse model of hus , neutralization of chemokines resulted in decreased renal fibrin deposition , suggesting that disrupting or eliminating these macrophage chemokines may be a useful treatment for the kidney damage associated with hus . currently , it is unclear whether il-8 and mcp-1 are induced in the cns . in the present study , we found that astrocytes were responsive to stx2 and produced il-8 and mcp-1 . in biological systems , further studies will need to address whether il-8 and mcp-1 are induced by stx2 in the cns , and what effects they exert .   no therapy for hus encephalopathy has yet been established . however , fujii et al . showed that betamethasone sodium phosphate pulse therapy reduced the mortality rate and prolonged the survival period of rabbits with stx2-toxemia . betamethasone sodium phosphate is routinely used as a steroid therapy for patients with brain edema . dexamethasone reduces the permeability of the normal bbb in mice . in brain astrocytes , kim et al . showed the coordinated regulation of angiotensin-1 and vascular endothelial growth factor ( vegf ) by dexamethasone , suggesting a novel mechanism underlying glucocorticoid - induced stabilization of the bbb . in addition , rock et al . examined cytokine and chemokine expression with and without dexamethasone in human microglia and astrocytes infected with mycobacterium tuberculosis . steroid protective effects are thought to be mediated by immunomodulatory activities , such as a decrease in the production of proinflammatory cytokines . therefore , anti - inflammatory compounds may have therapeutic potential for stx - induced neurological manifestation . investigations into the glucocorticoid regulation of chemokine expression by stx2 in hasts are currently underway . in summary , our results suggest a possible relationship between excessive chemokine production in the cns and brain damage once stx2 reaches the brain parenchyma . we propose that suppression of chemokine production could be a new strategy for treatment of cns damage in hus encephalopathy . in this study , we investigated the inflammatory responses in human astrocytes treated with stx2 . immunohistochemistry and real - time pcr revealed that the expression of globotriaosylceramide ( gb3 ) , the receptor for stx2 , and gb3 synthase ( galt6 ) in hasts was increased by interleukin-1 ( il-1 ) . chemokines in the cns may be associated with the pathogenesis of cns manifestations with hus .
infection with shiga toxin- ( stx- ) producing escherichia coli can lead to hemolytic uremic syndrome ( hus ) . 
 approximately , 30% of patients with hus suffer from complications in the central nervous system ( cns ) , which is an important determinant of mortality in such patients . 
 autopsy shows mostly edema and hypoxic - ischemic changes in the cns , often with microhemorrhages . 
 it has been suggested that stx - induced damage to human brain endothelial cells , which are essential constituents of the blood - brain barrier , plays a crucial role in the development of the cns complications . 
 however , it is unclear whether stx affects brain neuroglial cells . in the present study , we investigated the direct involvement of stx in the inflammatory responses of human astrocytes ( hasts ) treated with stx . 
 immunohistochemistry and real - time pcr revealed that the expression of globotriaosylceramide ( gb3 ) , the receptor for stx2 , and gb3 synthase ( galt6 ) in hasts was increased by interleukin-1 ( il-1 ) . 
 expression of both interleukin-8 ( il-8 ) and monocyte chemoattractant protein-1 ( mcp-1 ) mrna in hasts was significantly upregulated by stx2 . 
 these results suggest that stx2 induces inflammatory responses , particularly through expression of chemokines , in hasts expressing gb3 and may , thus , affect brain glial cells , playing a key role in the pathogenesis of cns manifestations associated with hus .