Escherichia coli Shiga toxin 1 and TNF-α induce cytokine release by human cerebral microvascular endothelial cells

Microbial Pathogenesis 36 (2004) 189–196 www.elsevier.com/locate/micpath

Escherichia coli Shiga toxin 1 and TNF-a induce cytokine release by human cerebral microvascular endothelial cells Patricia B. Eisenhauera,b,d, Mary S. Jacewiczd, Kelly J. Conna,b,c, Omanand Kould, John M. Wellsa,b, Richard E. Finea,b,c, David S. Newburgd,* a

Department of Veterans Affairs, VA Medical Center, Bedford, MA, USA Department of Neurology, Boston University School of Medicine, Boston, MA, USA c Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA d Program in Glycobiology, Shriver Center, University of Massachusetts Medical School, Waltham, MA, USA b

Received 4 July 2003; received in revised form 17 November 2003; accepted 19 November 2003

Abstract Infection with Shiga toxin (Stx)-producing Escherichia coli can lead to development of hemolytic uremic syndrome (HUS). Patients with severe HUS often exhibit central nervous system (CNS) pathology, which is thought to involve damage to brain endothelium, a component of the blood– brain barrier. We hypothesized that this neuropathology occurs when cerebral endothelial cells of the blood –brain barrier, sensitized by exogenous TNF-a and stimulated by Stx1, produce and release proinflammatory cytokines. This was tested by measuring changes in cytokine mRNA and protein expression in human brain endothelial cells (hBEC) in vitro when challenged by TNF-a and/or Stx. High doses of Stx1 alone were somewhat cytotoxic to hBEC; Stx1-treated cells produced increased amounts of IL-6 mRNA and secreted this cytokine. IL-1b and TNF-a mRNA, but not protein, were increased, and IL-8 secretion increased without an observed increase in mRNA. Cells pretreated with TNF-a were more sensitive to Stx1, displaying greater Stx1-induction of mRNA for TNF-a, IL-1b, and IL-6, and secretion of IL-6 and IL-8. These observations suggest that in the pathogenesis of HUS, Stx can induce cytokine release from hBEC, which may contribute toward the characteristic CNS neuropathology. q 2004 Published by Elsevier Ltd. Keywords: Escherichia coli; Shiga toxin; Hemolytic uremic syndrome; Brain endothelial cells; Cytokines

1. Introduction Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (STEC) infection is associated with bloody diarrhea and hemorrhagic colitis [1]. Hemolytic uremic syndrome (HUS) is most frequently the sequel of diarrhea due to infection by STEC. In the majority of HUS patients, damage is primarily concentrated in the renal endothelium. For patients with severe HUS, however, the endothelial damage extends to other organs, including the brain [2]. Up to 30% of HUS patients exhibit symptoms of central nervous system (CNS) pathology [1,3]. These patients have the poorest prognosis [4]. Stx crosses the intestinal mucosa and vascular endothelial layers, then binds with low affinity to blood cells, specifically * Corresponding author. Tel.: þ 1-781-642-0025; fax: þ1-781-642-0126. E-mail address: [email protected] (D.S. Newburg). 0882-4010/$ - see front matter q 2004 Published by Elsevier Ltd. doi:10.1016/j.micpath.2003.11.004

polymorphonuclear leukocytes (PMNs) and monocytes and may therefore be transported to the brain by these cells. Stx2 has been reported to be bound to PMNs of patients during the acute phase of HUS, concurrent with the presence of bloody diarrhea [5]. Although tissue damage in HUS results from direct cytotoxic effects of Stx on vascular endothelium [2], Stx alone cannot account for all the clinical manifestations of HUS neuropathology. A possible role of cytokines in the pathogenesis of the disease has also been suggested. Infection by STEC leads to the production of cytokines in the intestine, which reach the brain and other distal sites via the systemic circulation [6]. Clinical studies have demonstrated that HUS patients have elevated levels of cytokines, including TNF-a and IL-1b, in plasma as well as in urine; IL-6 is elevated in the serum of HUS patients [7]. A healthy blood –brain barrier is essential to protect CNS function. Tight junctions of brain endothelial cells (BEC)

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are an essential component of the blood – brain barrier, making it resistant to passage of macromolecules. Elevated cytokine levels have been implicated in the disruption of blood – brain barrier function in inflammatory states: For example, TNF-a, IL-1b, and IL-6 are increased in blood and edema fluid after tissue injury [8]. BEC are exposed to circulating Stx and proinflammatory cytokines that are elevated in HUS. The role of cytokines in the pathogenesis of the neurological manifestations of HUS, however, is not understood. Our hypothesis is that an inflammatory response to Stx in human BEC (hBEC) elicits local cytokine production that contributes to the neuropathology of HUS. We studied the effect of Stx1 and TNF-a on the cytokine production and cell viability of hBEC. The ability of Stx to induce cytokine mRNA, cause release of cytokine into the medium, or alter

intracellular cytokine levels was measured in basal hBEC and hBEC treated with TNF-a.

2. Results 2.1. Induction of cytokine mRNAs in hBEC Fig. 1 shows a typical result for cells treated with 1027 and 10 M Stx1 with and without pretreatment with TNF-a. 28

(a) TNF-a mRNA, IL-1b mRNA, and IL-6 mRNA were induced in cells treated with Stx1 alone. The increase in IL-1b mRNA in untreated cells (Fig. 1B) was statistically significant in hBEC treated with 1027 M, but not 1028 M, Stx1. The increases in TNF-a mRNA

Fig. 1. Stx1 induction of cytokine mRNAs in hBEC cultures. Expression of cytokine mRNAs was detected by RT-PCR using RNA extracted from hBEC treated with TNF-a (100 U/ml), Stx1 (1027 or 1028 M), or both for 6 h as described in Section 4. G3PDH was used as a control for total RNA. Data are expressed as % mRNA (mean ^ SD) of control cultures that had not been treated with Stx1 or TNF-a. (A). TNF-a; (B). IL-1b; (C). IL-6; (D). IL-8. The results are from a representative experiment of at least three replicate experiments. Statistics: Data were analyzed for comparison of multiple means by one-way ANOVA followed by Tukey–Kramer test. Comparisons were made between Stx1 and control (0); TNF-a and control; TNF-a and TNF-a þ Stx1; and Stx1 and TNF-a þ Stx1 for all doses of Stx1. Significant P values: TNF-a: TNF-a vs. TNF-a þ 1027 M Stx1, P , 0:01; TNF-a vs. TNF-a þ 1028 M Stx1, P , 0:05; 1027 M Stx1 vs. TNF-a þ 1027 M Stx1, P , 0:01. IL-1b: control vs. 1027 M Stx1, P , 0:01; control vs. TNF-a, P , 0:05; TNF-a vs. TNFa þ 1027 M Stx1, P , 0:01; TNF-a vs. TNF-a þ 1028 M Stx1, P , 0:05; 1028 M Stx1 vs. TNF-a þ 1028 M Stx1, P , 0:01. IL-6: control vs. TNF-a, P , 0:05; TNF-a vs. TNF-a þ 1027 M Stx1, P , 0:05; 1027 M Stx1 vs. TNF-a þ 1027 M Stx1, P , 0:01. All other comparisons were not significant ðP . 0:05Þ.

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(Fig. 1A) and IL-6 mRNA (Fig. 1C) induction were not statistically significant, and no IL-8 mRNA induction was observed (Fig. 1D). (b) Pretreatment of hBEC with TNF-a alone (0 Stx1) caused a significant increase relative to untreated hBEC in IL-1b mRNA (Fig. 1B) and IL-6 mRNA (Fig. 1C), but not TNF-a mRNA (Fig. 1A) or IL-8 mRNA (Fig. 1D). (c) TNF-a and Stx1 in combination were synergistic in their induction of some cytokine mRNAs. TNF-a mRNA levels (Fig. 1A) in hBEC exposed to TNF-a and 1027 M Stx1 were higher than in cells treated with either TNF-a alone or 1027 M Stx1 alone. In cells exposed to TNF-a and 1028 M Stx1 the increase was significant only when compared with TNF-a-treated cells, but not when compared with cells treated with 1028 M Stx1 alone. The induction of IL-1b mRNA (Fig. 1B) was statistically significant at both 1027 and 1028 M Stx1 in hBEC pretreated with TNF-a compared with cells treated with TNF-a alone, but was significant only in cells treated with 1028 M Stx1 when compared with cells treated with the corresponding dose of toxin alone. IL-6 mRNA induction in hBEC (Fig. 1C) treated with both TNF-a and 1027 M Stx1 was greater than in cells treated with either TNF-a alone or 1027 M Stx1 alone, but no significant increase was observed in cells treated with TNF-a and 1028 M Stx1. No changes were observed in IL-8 (Fig. 1D) production by hBEC treated with Stx1 and TNF-a. Our findings showed that the greatest induction of cytokine mRNAs occurred in hBEC treated with the combination of TNF-a and 1027 M Stx1. There was no evidence for toxin-related changes in mRNA for any of the cytokines in cells treated with 1029 M Stx1 alone or in combination with TNF-a. 2.2. Cytokine synthesis by hBEC in reponse to Stx To determine whether the upregulation of the mRNA for the cytokines was accompanied by release of these cytokines into the medium, hBEC were exposed to Stx1

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for 6, 24, or 48 h and the concentration of cytokines was measured in the medium. (a) Treatment of hBEC with 1027 to 1029 M Stx1 alone resulted in no significant change in TNF-a secretion at any time point tested (data not shown). Treatment with 1027 M Stx1 increased secretion of IL-6 (Table 1) above untreated control levels; this increase was small but significant at 6 h, rose to 2-fold at 24 h, and to 3fold at 48 h. Stx1 at 1028 or 1029 M did not result in increased IL-6 levels at 6 h, but IL-6 levels increased significantly after 24- and 48-h treatment at these concentrations of toxin. Treatment with 1027 M Stx1 for 6 h increased IL-8 secretion 3-fold (Table 2), even though IL-8 mRNA was not affected. Exposure of cells to 1028 and 1029 M Stx1 caused a slight but significant increase in IL-8 at 6 h and a larger increase at 24 h; however, IL-8 levels returned to control values by 48 h (Table 2). (b) TNF-a (100 U/ml) treatment of hBEC resulted in increased levels of both IL-6 and IL-8 at all time points tested. For IL-6 (Table 1), the increase was 16-fold at 6 h, rising to 46-fold at 24 h, and 38-fold at 48 h, compared with the levels in untreated cells at the corresponding time point. For IL-8 (Table 2), the increase was 3-fold at 6 h, rising to 6-fold at 24 h, and 4-fold at 48 h, compared with the levels in untreated cells at the corresponding time point. For both IL-6 and IL-8, the increase relative to controls was less at 48 than at 24 h because in untreated cells there was a greater relative increase in secretion of cytokines into the supernatant between 24 and 48 h than in treated cells. (c) Most noteworthy are the increases in both IL-6 and IL8 levels that occurred in TNF-a primed hBEC treated with Stx. These increases were significant ðP , 0:01Þ at all times and toxin doses tested relative to levels in cells treated with Stx1 alone. TNF-a plus Stx1 (1027 M) for 6, 24, and 48 h resulted in a significant increase in secreted IL-6 (Table 1), compared with cells treated with TNF-a alone, while at 1028 and 1029 M

Table 1 IL-6 levels in culture media of untreated or TNF-a-treated hBEC exposed to Stx1 (Stx1)M

IL-6 (pg/mL) þ TNF-a (h)

No TNF-a (h)

0 1029 1028 1027

6

24

48

6

24

48

55 ^ 4 54 ^ 5 (ns) 52 ^ 2 (ns) 78 ^ 6 (,0.01)

48 ^ 1 101 ^ 3 (,0.01) 92 ^ 9 (,0.01) 107 ^ 18 (,0.01)

74 ^ 2 116 ^ 9 (,0.01) 98 ^ 8 (,0.05) 207 ^ 12 (,0.01)

852 ^ 21 831 ^ 72 (ns) 816 ^ 24 (ns) 1008 ^ 62 (,0.01)

2207 ^ 158 3019 ^ 117 (,0.01) 2832 ^ 29 (,0.01) 3618 ^ 429 (,0.01)

2786 ^ 258 3150 ^ 108 (ns) 2938 ^ 287 (ns) 4385 ^ 468 (,0.01)

Data are the mean ^ SD of triplicate data points from one representative experiment. Similar results were obtained for at least four experiments. P-values in parentheses measure differences between cells treated with Stx (1029, 1028, or 1027 M Stx1) and those treated identically but without addition of Shiga toxin (0 Stx1). Data were analyzed for comparison of multiple means by one-way ANOVA followed by Tukey–Kramer test.

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Table 2 IL-8 levels in culture media of untreated or TNF-a-treated hBEC exposed to Stx1 [Stx1]M

IL-8 (ng/ml) þTNF-a (h)

No TNF-a (h)

0 1029 1028 1027

6

24

48

6

24

48

6.2 ^ 0.4 7.7 ^ 0.2 (,0.05) 7.8 ^ 0.5 (,0.05) 19.1 ^ 0.8 (,0.01)

10.9 ^ 0.8 14.0 ^ 0.6 (,0.05) 21.8 ^ 0.9 (,0.01) 23.6 ^ 1.1 (,0.01)

23 ^ 1 24 ^ 2 (ns) 23 ^ 1 (ns) 58 ^ 4 (,0.01)

21.6 ^ 1.1 28.0 ^ 3.2 (,0.01) 19.9 ^ 1.3 (ns) 24.8 ^ 0.9 (ns)

71 ^ 2 176 ^ 4 (,0.01) 119 ^ 3 (,0.01) 119 ^ 2 (,0.01)

97 ^ 9 222 ^ 7 (,0.01) 194 ^ 5 (,0.01) 208 ^ 6 (,0.01)

Data are the mean ^ SD of triplicate data points from one representative experiment. Similar results were obtained for at least four experiments. P-values in parentheses measure differences between cells treated with Stx (1029, 1028, or 1027 M Stx1) and those treated identically but without addition of Shiga toxin (0 Stx1). Data were analyzed for comparison of multiple means by one-way ANOVA followed by Tukey–Kramer test.

Stx1, the only significant changes occurred at 24 h. Incubation of TNF-a pretreated hBEC with 1029 M Stx1 for 6 h resulted in significantly increased IL-8 levels (Table 2), whereas the higher concentrations of toxin had no effect. TNF-a pretreated hBEC incubated for 24 or 48 h with Stx1 secreted significantly more IL8 at all toxin concentrations tested than untreated controls. No evidence was found for Stx-induced secretion of IL-1b, with or without TNF-a pretreatment (data not shown), despite the induction of IL-1b mRNA. TNF-a pretreatment of cells (100 U/ml for 48 h) did not change the minimal effect of Stx1 on secretion of TNF-a. Indeed, TNF-a levels decreased over time at all doses tested (data not shown). (d) Cytokine levels in lysates of cells exposed to Stx1 for 24 or 48 h were also measured. These intracellular levels were low and were not changed by TNF-a or by Stx1 treatment, with or without TNF-a pretreatment, suggesting that any cytokines produced may have been secreted (data not shown) rather than stored. (e) The levels of mRNA expression and cytokine production varied somewhat between experiments. These differences appeared to depend on the passage number and length of time that the cells were stored frozen. Cells that were frozen for relatively long periods expressed less TNF-a mRNA following Stx1 treatment than did cells that had not been frozen or had been frozen for a short period of time. This could be due to the changes in the rate of transcription or in the stability of the message; we did not specifically address this issue. All data presented herein were reproducible in at least three or four independent experiments.

dilutions of toxin. Pretreatment of cells with 100 U/ml TNF-a for 48 h did not increase the sensitivity of the cells to treatment with Stx1 for 6 h (not shown), but pretreatment of these cells with TNF-a increased their sensitivity to Stx1 after 24 h of exposure. The results for 24 h of toxin exposure (Fig. 2) were consistent with the previously reported study [9].

2.3. Cytotoxicity of Shiga toxin

Fig. 2. Cytotoxic effect of Shiga toxin 1 on hBEC. hBEC were preincubated with medium alone or with medium containing 100 U/ml TNF-a for 48 h before addition of Stx1 (24-h exposure). Viability was determined by MTT assay. Control cells incubated in medium alone were used as the basis for calculating percent viability ðn ¼ 6 for each data point) and data were expressed as mean ^ standard deviation. Comparisons were made between Stx1-induced toxicity and Stx1 þ TNF-a-induced toxicity. P values for all doses at this time point are , 0:05.

In a previous study, hBEC displayed a time- and dosedependent decrease in cell viability for 6- and 24-h incubation periods at Stx1 concentrations ranging from 1029 to 1027 M [9]. We found no evidence of toxicity of Stx1 to hBEC after 6 h of exposure to serial 10-fold

3. Discussion CNS pathology is an important complication of epidemic HUS, but little is known about how these symptoms arise. Stx and proinflammatory cytokines have been implicated in the pathogenesis of this disease. Following STEC infection, circulating toxin and cytokines can affect the hBEC of the blood –brain barrier. This barrier, responsible for maintaining homeostasis within the CNS, is a site at which important molecular transport between blood and the brain is controlled. Although neutrophils are a major source of

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proinflammatory cytokines, endothelium-derived cytokines may also contribute to alterations in the integrity of the blood – brain barrier. The cells comprising the endothelial monolayer of the blood – brain barrier are resistant to Stx in vitro. However, endothelial cells can be sensitized to Stx by the presence of circulating proinflammatory cytokines, including TNF-a and IL-1b [9,10] by increasing the level of globotriaosylceramide (Gb3), the receptor for Stx in BEC. Furthermore, BEC, when activated by TNF-a, can trigger a series of inflammatory reactions that could result in increased levels of cytokines bathing the glia and neurons of the CNS, and may ultimately lead to neural cell death and other CNS disruptions of the type that can occur in HUS [6,11]. The studies described herein demonstrate that hBEC in vitro produce cytokines in response to Stx1. Elevated levels of mRNA for IL-1b, TNF-a, and IL-6 were found in toxintreated cells compared with untreated controls; this mRNA expression was further increased when cells were pretreated with TNF-a. IFN-g mRNA and protein secretion were unaltered by either TNF-a, Stx1, or both, at the doses tested (data not shown). IL-8 and IL-6 proteins were found at higher levels in media of Stx1-treated cells than in untreated cells even though IL-8 mRNA was not increased by toxin treatment. IL-1b protein did not increase, despite the elevated levels of IL-b mRNA. Most importantly, TNF-a pretreatment of cells prior to Stx1 exposure resulted in higher IL-6 and IL-8 cytokine levels than in cells treated with toxin or TNF-a alone, but did not induce production of IL-1b. A precursor form of IL-1b may be sequestered within intracellular stores and release of bioactive IL-1b may require the activation of the cysteine proteinase IL-1b converting enzyme (caspase-1) [12]. Increased levels of cytokines were found in the cell media, but not in the cell lysates, indicating that cytokines were secreted. Our results agree with a previous report in which hBEC treated with Stx1 for 18 h produced elevated IL-6, but only trace amounts of TNF-a, and no IL-1b [10]. Stx1 treatment has been shown to increase expression of cytokine mRNA and protein in various cell types. Both mRNA and protein secretion of IL-1b, IL-6, and TNF-a were increased in human glomerular endothelial cells treated with Stx1 and lipopolysaccharide [13]. Increases in TNF-a, IL-1b, IL-6, and IL-8 protein and a parallel increase in IL-6 mRNA have been reported in Stx1-treated peripheral blood monocytes. Increases in TNF-a and IL-1b production followed Stx1 treatment of peripheral blood monocytes and differentiated monocytic cell lines. Stx-induced superinduction of IL-8 mRNA and protein, despite a general shutdown of mRNA translation, has also been demonstrated in human colonic epithelial cell lines HCT-8 [14,15] and CaCo2 [16]. Cytokines in the presence of Stx may be due to a ribotoxic stress response, a generalized cellular response to molecules that damage 28S rRNA. Our findings suggest a mechanism whereby Stx could damage the blood – brain barrier, and enable access of

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factors to neural cells that cause the neuropathological manifestations of HUS. This leads us to propose the following model for the pathogenesis of cerebral damage due to HUS. Stx, lipopolysaccharides, and proinflammatory cytokines are produced and released into the intestinal lumen during infection with STEC. Stx crosses intestinal epithelial and endothelial cell layers and enters the blood stream, where it binds to blood cells and circulates to the brain microvasculature together with the proinflammatory cytokines released at the locus of infection. The cytokines activate the brain endothelium, which in turn produces a variety of factors, including cytokines and adhesion factors [17]. Expression of adhesion factors on the endothelial surface renders it sticky to leukocytes, which attach to the surface and may thus cross the blood –brain barrier. At the same time, TNF-a and IL-1b sensitize the endothelium to Stx by upregulating the Stx receptor, Gb3, on the cell surface [9,18]. After binding to the endothelium, Stx enters the cells, inhibits total protein synthesis, and triggers production of IL-6 and IL-8. Stx toxicity and upregulation of cytokines disrupt tight junctions, and facilitate crossing of the blood – brain barrier by electrolytes, leukocytes, Stx, lipopolysaccharides, cytokines, and other serum components. Astrocytes and microglia, which are also components of the blood – brain barrier, respond to injury by releasing cytokines, including TNF-a, IL-6, and IL-8 [19]. These inflammatory mediators released by the cells of the blood – brain barrier, together with incoming leukocytes, and factors released by them, initiate a series of signaling cascades ultimately contributing to the damage to brain parenchyma that is observed in HUS. In summary, our data demonstrate that hBEC respond to Stx1 by secreting increased amounts of cytokines that may potentiate toxin-induced damage to the cells of the blood – brain barrier. Thus, many of the neurological complications of HUS could be accounted for by the synergy between the effects of Stx and cytokines on hBEC.

4. Materials and methods 4.1. Toxin purification Stx1 was purified from cell lysates of E. coli HB101H19B, an STEC expressing Stx1 only. The toxin was purified by affinity chromatography on a P1 blood group glycoprotein – Sepharose 4B column, as previously described [20], resulting in a preparation yielding two bands on SDS PAGE, corresponding to the A and B subunits. 4.2. Brain endothelial cell culture hBEC cells were isolated from human cerebral cortex obtained from autopsy as previously described [21]. Briefly, the brain sample was cleaned of meninges and associated

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surface vessels; cortical grey matter was dissected, homogenized, and filtered, and the resulting microvessel fraction was digested with collagenase/dispase (1 mg/ml) and DNAse (10 mg/ml) for 1 h at 37 8C. Following digestion, the sample was centrifuged, and the microvessels were suspended in a medium consisting of 50% MEM containing 10% plasma-derived horse serum, 50% astrocyte-conditioned medium, and 1.25 ng/ml bFGF supplement (Sigma, St. Louis, MO). The astrocyte-conditioned medium was prepared essentially as described by Rubin et al. [22]. Cells were characterized on the basis of morphology and the presence of Factor VIII (95% positive). Cells were used between passages 2 through 4.

4.4. RNA isolation and reverse transcription Total cellular RNA was isolated using Tri Reagent (Sigma) according to the manufacturer’s instructions. Approximately 50 mg of RNA was treated with 1 U DNAse-I using the Message Clean kit (GenHunter, Nashville, TN). DNAse-treated RNA was subjected to PCR analyses as described below, using primers to glyceraldehyde-3-phosphate dehydrogenase (G3PDH; EC 1.2.1.12) to ensure that each RNA preparation was free of DNA. DNAse-treated RNA (5 mg) was reverse transcribed using oligo (dT) primers provided in the Superscript kit (Clontech, Palo Alto, CA).

4.3. Experimental design 4.5. PCR amplification, visualization and quantitation The effects of exogenous Stx1 and/or TNF-a on hBEC monolayers were investigated with regard to their effects on cell viability, and on induction of mRNA for various cytokines (listed below), as well as on expression of cytokine proteins. The effect of TNF-a on sensitivity of hBEC to Stx1 and any synergistic effects of TNF-a and Stx1 on cytokine mRNA and protein production were investigated by preincubating cells with TNF-a for 48 h prior to the addition of toxin. 4.3.1. Sensitivity of hBEC to Stx1 hBEC were preincubated with medium alone or with medium containing 100 U/ml TNF-a for 48 h before addition of serial 10-fold dilutions of Stx1 (1027 to 1029 M, for 6 or 24 h). Viability was determined by the MTT assay, described below. Control cells incubated in medium alone were used to calculate percent viability (n ¼ 6 for each data point) and data were expressed as mean ^ standard deviation. 4.3.2. Induction of cytokines by Stx1 and TNF-a Confluent cultures of hBEC, with or without 48-h pretreatment with 100 U/ml TNF-a, were exposed to serial 10-fold dilutions of Stx1 (1029 to 1027 M) for 6 h. Cytokine mRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR), described below. 4.3.3. Detection of cytokine proteins hBEC cultures pretreated with 100 U/ml TNF-a or with medium alone for 48 h were exposed to various concentrations of Stx1 for 6, 24, or 48 h. The culture supernatants were collected by centrifugation and analyzed immediately or stored at 2 70 8C. For cell-associated cytokine analysis, 500 ml of fresh medium was added to the cells, which were subjected to three cycles of freezing and thawing. Samples were centrifuged before assay. Cytokine proteins were detected by enzyme-linked immunosorbent assay (ELISA), described below.

Conditions were optimized for each primer set to provide data in the linear range for the PCR. PCR reactions were performed in a final volume of 25 ml containing 1 ml of cDNA product, 2.5 ml of 10 £ reaction buffer (Perkin Elmer/Roche, Branchburg, NJ), 0.2 mM dNTP product (Invitrogen; Carlsbad, CA), 1 unit Taq polymerase (Perkin Elmer/Roche), and 0.5 mg each of 30 and 50 primer DNA. Primers for the amplification of IFNg were purchased from Clontech. Primers for the amplification of G3PDH, IL-6, IL-8, TNF-a, and IL-1b were purchased from Invitrogen Life Technologies (Carlsbad, CA). These primer sequences were originally obtained from Clontech (Palo Alto, CA). Polymerase chain reaction products were resolved by electrophoresis on 2% agarose gels containing 0.5 mg/ml ethidium bromide. Resolved PCR products were visualized using the 4400 ChemiImager low-light imaging system (Alpha-Innotech; San Leandro, CA). Quantitation was by spot densitometry of the electronically captured images. The optimal number of PCR cycles for each primer set is given in Table 3. To minimize the possibility that the PCR products shown in this study are the result of amplification of small amounts of gene transcripts from non-hBEC cells, the following steps were taken: To ensure the estimation of gene expression is quantitative, a sophisticated imaging system was used to capture and quantify images of PCR products separated by agarose gel electrophoresis that allowed quantification of PCR products within the linear range of detection for the instrument. PCR products generated from increasing numbers of PCR cycles were quantified and the spot densitometry values for each of these products was graphed against the cycle number to determine the number of PCR cycles needed to be within the linear range for the PCR for each primer set. For the majority of genes tested in this study (including the ubiquitous housekeeping gene G3PDH), 30 PCR cycles were optimal.

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Table 3 Primers, conditions, and optimal number of cycles used for RT-PCR analyses of cytokine mRNA expressed by hBEC Name

Primer sets

# of PCR cycles

PCR product (bp)

G3PDH

50 -ACCACAGTCCATGCCATCAC-30 50 -TCCACCACCCTGTTGCTGTA-30 50 -ATGAACTCCTTCTCCACAAGCGC-30 50 -GAAGAGCCCTCAGGCTGGACTG-30 50 -ATGACTTCCAAGCTGGCCGTGGCT-30 50 -TCTCAGCCCTCTTCAAAAACTTCTC-30 50 -GAGTGACAAGCCTGTAGCCCATGTTGTAGA-30 50 -GCAATGATCCCAAAGTAGACCTGCCCAGACT-30 50 -ATGGCAGAAGTACCTAAGCTCGC-30 50 -ACACAAATTGCATGGTGAAGTCAGTT-30 50 -GCATCGTTTTGGGTTCTCTTGGCTGTTACTC-30 50 -CTCCTTTTTCGCTTCCCTGTTTTAGCTGCTGG-30

30

450

35

628

30

289

35

444

35

802

30

427

IL-6 IL-8 TNF-a IL1-b INF-g

PCR cycle: 94 8C, 1 min; 55 8C, 1 min; 72 8C, 2 min. The same cycle was used for each set of primers.

4.6. Detection of cytokine proteins by enzyme-linked immunosorbent assay (ELISA) Culture supernatants from treated hBEC were collected by centrifugation and analyzed immediately or stored at –70 8C. For cell-associated cytokine analysis, 500 ml of fresh medium was added to the cells, which were subjected to three cycles of freezing and thawing. Samples were centrifuged before assay. The ELISAs for IL-1b, IL-6, IL-8, TNF-a, and IFN-g were performed using commercial ELISA kits (Biosource International, Camarillo, CA) according to manufacturers’ instructions. Briefly, culture supernatants were incubated in the specific-capture antibody-coated wells, and bound cytokine was detected by incubation with a biotin-labeled detection antibody followed by horseradish peroxidase-conjugated streptavidin and tetramethylbenzidine chromogen. Cytokines were quantified by measurement of A450 on a BT2000 Microkinetics Reader (Bio-Tek Instruments, Inc., Winooski, VT) and the concentration of the cytokine in the sample was determined from a standard curve. 4.7. Cytotoxicity assay The MTT assay was used to assess cell viability. This assay measures the reduction of 3-(4,5-dimethylthiazol2yl)-2,5-diphenyltetrazolium bromide (MTT) to a colored formazan derivative via mitochondrial dehydrogenase activity. The assay was performed according to the manufacturer’s instructions (Promega, Madison, WI). In brief, MTT was added to treated hBEC, and absorbances were read at 570 nm with an automated plate reader. Results were expressed as percent reduction of MTT uptake relative to controls. 4.8. Statistics The data were analyzed by Student’s t-test for independent samples. When appropriate, data were analyzed for

comparison of multiple means by one-way ANOVA followed by the Tukey – Kramer test.

Acknowledgements Presented in part: 100th General Meeting of the American Society for Microbiology, Los Angeles, CA, May 23, 2000 (abstract D172). The primary BEC cultures were obtained from the Boston University Alzheimer’s Disease Center Neuropathology Core, an NIH-funded repository. Financial support: Merit Review Entry Program (MREP) and Merit Review grants and a Research Enhancement Award Program (REAP) grant from the Medical Research Service, Department of Veterans Affairs Medical Center, Bedford, MA. Dr P. Eisenhauer is a MREP awardee. Dr K. Conn is a REAP fellow. National Institutes of Health Grants DK52122, DK59811, AG13846, and P30 DK34928 for the Center for Gastroenterology Research on Absorptive and Secretory Processes. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the awarding agencies. We thank Ms Kathryn Newburg and Ms Louise Kittredge and for expert editorial assistance.

References [1] Lopez EL, Diaz M, Grinstein S, Devoto S, Mendilaharzu F, Murray BE, et al. Hemolytic uremic syndrome and diarrhea in Argentine children: the role of Shiga-like toxins. J Infect Dis 1989;160:469– 75. [2] Kaplan BS, Cleary TG, Obrig TG. Recent advances in understanding the pathogenesis of the hemolytic uremic syndromes. Pediatr Nephrol 1990;4:276 –83. [3] Sheth KJ, Swick HM, Haworth N. Neurological involvement in hemolytic-uremic syndrome. Ann Neurol 1986;19:90–3. [4] Mariani-Kurkdjian P, Bingen E. Hemolytic-uremic syndrome after verotoxin-producing Escherichia coli infection. Presse Med 1995;24: 99–101. [5] Te Loo DM, van Hinsbergh VW, van den Heuvel LP, Monnens LA. Detection of verocytotoxin bound to circulating polymorphonuclear

196

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

P.B. Eisenhauer et al. / Microbial Pathogenesis 36 (2004) 189–196 leukocytes of patients with hemolytic uremic syndrome. J Am Soc Nephrol 2001;12:800–6. Isogai E, Isogai H, Kimura K, Hayashi S, Kubota T, Fujii N, et al. Role of tumor necrosis factor alpha in gnotobiotic mice infected with an Escherichia coli O157:H7 strain. Infect Immun 1998;66:197–202. Karpman D, Andreasson A, Thysell H, Kaplan BS, Svanborg C. Cytokines in childhood hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Pediatr Nephrol 1995;9:694–9. Ali MH, Schlidt SA, Chandel NS, Hynes KL, Schumacker PT, Gewertz BL. Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction. Am J Physiol 1999;277: L1057–65. Eisenhauer PB, Chaturvedi P, Fine RE, Ritchie AJ, Pober JS, Cleary TG, et al. Tumor necrosis factor alpha increases human cerebral endothelial cell Gb3 and sensitivity to Shiga toxin. Infect Immun 2001;69:1889 –94. Ramegowda B, Samuel JE, Tesh VL. Interaction of Shiga toxins with human brain microvascular endothelial cells: cytokines as sensitizing agents. J Infect Dis 1999;180:1205–13. Fujii J, Kita T, Yoshida S, Kakeda T, Kobayashi H, Nakata N, et al. Direct evidence of neuron impairment by oral infection with verotoxin-producing Escherichia coli 0157:H- in mito-mycin-treated mice. Infect Immun 1994;62:3447–53. Wewers MD, Winnard AV, Dare HA. Endotoxin-stimulated monocytes release multiple forms of IL-1 beta, including a proIL-1 beta form whose detection is affected by export. J Immunol 1999;162: 4858–63. Hughes AK, Stricklett PK, Kohan DE. Shiga toxin-1 regulation of cytokine production by human glomerular epithelial cells. Nephron 2001;88:14 –23.

[14] Thorpe CM, Hurley BP, Lincicome LL, Jacewicz MS, Keusch GT, Acheson DW. Shiga toxins stimulate secretion of interleukin-8 from intestinal epithelial cells. Infect Immun 1999;67:5985– 93. [15] Smith WE, Kane AV, Campbell ST, Acheson DW, Cochran BH, Thorpe CM. Shiga toxin 1 triggers a ribotoxic stress response leading to p38 and JNK activation and induction of apoptosis in intestinal epithelial cells. Infect Immun 2003;71:1497– 504. [16] Yamasaki C, Natori Y, Zeng XT, Ohmura M, Yamasaki S, Takeda Y. Induction of cytokines in a human colon epithelial cell line by Shiga toxin 1 (Stx1) and Stx2 but not by non-toxic mutant Stx1 which lacks N-glycosidase activity. FEBS Lett 1999;442:231 –4. [17] Stanimirovic D, Satoh K. Inflammatory mediators of cerebral endothelium: a role in ischemic brain inflammation. Brain Pathol 2000;10:113–26. [18] Keusch GT, Acheson DW, Aaldering L, Erban J, Jacewicz MS. Comparison of the effects of Shiga-like toxin 1 on cytokine- and butyrate-treated human umbilical and saphenous vein endothelial cells. J Infect Dis 1996;173:1164–70. [19] Aschner M. Immune and inflammatory responses in the CNS: modulation by astrocytes. Toxicol Lett 1998;102-103:283–7. [20] Donohue-Rolfe A, Jacewicz M, Keusch GT. Isolation and characterization of functional Shiga toxin subunits and renatured holotoxin. Mol Microbiol 1989;3:1231–6. [21] Eisenhauer PB, Wells JM, McKenna DC, Shanmugaratnam J, Fine RE, Billingslea AM, et al. b-APP processing and regulation by interleukin 1 in brain endothelial cells. Alzheimer’s Rep 1998;1: 241 –7. [22] Rubin L, Hall D, Porter S, Barbu K, Cannon C, Horner HC, et al. A cell culture model of the blood brain barrier. J Cell Biol 1991;115: 1725–35.