Elsevier

Microbial Pathogenesis

Volume 36, Issue 4, April 2004, Pages 189-196
Microbial Pathogenesis

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

https://doi.org/10.1016/j.micpath.2003.11.004Get rights and content

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-α 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-α 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-1β and TNF-α mRNA, but not protein, were increased, and IL-8 secretion increased without an observed increase in mRNA. Cells pretreated with TNF-α were more sensitive to Stx1, displaying greater Stx1-induction of mRNA for TNF-α, IL-1β, 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.

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 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-α and IL-1β, 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) 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-α, IL-1β, 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-α 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-α.

Section snippets

Induction of cytokine mRNAs in hBEC

Fig. 1 shows a typical result for cells treated with 10−7 and 10−8 M Stx1 with and without pretreatment with TNF-α.

  • (a)

    TNF-α mRNA, IL-1β mRNA, and IL-6 mRNA were induced in cells treated with Stx1 alone. The increase in IL-1β mRNA in untreated cells (Fig. 1B) was statistically significant in hBEC treated with 10−7 M, but not 10−8  M, Stx1. The increases in TNF-α mRNA (Fig. 1A) and IL-6 mRNA (Fig. 1C) induction were not statistically significant, and no IL-8 mRNA induction was observed (Fig. 1D).

  • (b)

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

Toxin purification

Stx1 was purified from cell lysates of E. coli HB101-H19B, 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.

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 surface

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

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