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Is immune cell activation the missing link in the pathogenesis of post-diarrhoeal HUS?
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Opinion
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Is immune cell activation the missing link in the pathogenesis of postdiarrhoeal HUS? Robert S. Heyderman, Marco Soriani and Timothy R. Hirst Haemolytic uraemic syndrome (HUS), which is caused by Shiga toxin (Stx)producing Escherichia coli, is the commonest cause of acute renal failure in childhood. It is widely believed that HUS develops following the release of Stx, an AB5 toxin that inhibits protein synthesis and has a direct toxic effect on the kidney endothelium. There remains, however, a mismatch between the current understanding of the pathogenesis of HUS and the evolution of the clinical signs of the disease. Our hypothesis is that Stx-mediated immune cell activation in the gut is the missing link in the pathogenesis of this condition, initiating the characteristic renal pathology of HUS either alone or in synergy with Stx. Validation of this hypothesis could lead to a targeted anti-inflammatory approach aimed at modulating immune cell function in HUS.
Robert S. Heyderman* Marco Soriani Timothy R. Hirst Dept of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, UK BS8 1TD. *e-mail: r.heyderman@ bristol.ac.uk
In the past decade, multiple outbreaks of Shiga toxin (Stx)-producing Escherichia coli O157:H7mediated gastroenteritis and haemolytic uraemic syndrome (HUS) have highlighted the global public health impact of this emerging pathogen1–3. HUS occurs in ~5% of E. coli O157:H7-infected individuals with bloody diarrhoea and is characterized by acute renal failure, thrombocytopaenia and microangiopathic haemolytic anaemia. The precise sequence of events leading to the development of HUS is unknown3,4. Although it is widely believed that Stx exerts a direct toxic effect on the endothelium of the kidney, recent evidence suggests that AB5 toxins, particularly the B subunit, have many biological effects and can activate signal transduction pathways, thereby influencing epithelial and leukocyte survival, activation and cell death5,6. These new insights have led us to propose a novel immune-mediated mechanism for the development of E. coli O157:H7-mediated HUS. Stx and pathogenesis
The E. coli Stx family of toxins has two major members: Stx1, which structurally is virtually identical to the Shiga toxin produced by
Shigella dysenteriae type 1, and Stx2, which has only 56% homology to Stx1. All Stx family members have an A subunit (StxA) with RNA-glycohydrolase activity and five B subunits (StxB), which bind with high affinity to the glycolipid receptor Gb3 (CD77) found on the surface of eukaryotic cells7. StxA alone is unable to initiate toxicity because it cannot bind to the Gb3 cell surface receptor. Receptor binding by the holotoxin triggers Stx uptake and trafficking to the endoplasmic reticulum, leading to translocation of the enzymatic A subunit into the cytosol where it catalyses depurination of a single adenine residue in the 28S rRNA of 60S ribosomes, resulting in inhibition of protein synthesis7. Stx also triggers signalling events that lead to apoptosis in endothelial and epithelial cells6,8–10, activation of the Src kinase family11 and nuclear translocation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1)12. Although the contribution of the toxic A subunit to the induction of these events has been demonstrated, it is also clear that the B subunit possesses signalling properties in its own right that can cause apoptosis of cells expressing the Gb3 receptor5,6.
‘...mechanisms other than endothelial apoptosis are important in the initiation of the microvascular thrombosis associated with HUS.’ Limitations of Stx as the sole mediator of HUS
Several features of HUS suggest that Stx-mediated toxicity alone is not sufficient to explain the epidemiological link between Stx and the development of HUS. First, if the glomerular lesions are toxin mediated, why is there a delay of five to seven days between the onset of diarrhoea, when E. coli O157:H7 and its toxin are most easily detected in the stool13,14, and the subsequent development of HUS? It has been suggested that this delay represents the time it takes for Stx to accumulate at its target sites or the time taken for the toxin to induce clinical disease15. However, these explanations fail to account for the poor correlation between the level of intestinal toxin, the clinical outcome of individual patients13 and the abrupt nature of the host inflammatory response associated with HUS. Second, how does Stx, a potent inhibitor of protein synthesis, trigger such a marked inflammatory process with cytokine production and widespread neutrophil activation? Alternative signalling pathways must be involved in the inflammatory response associated with the disease16. Finally, how is the glomerular thrombosis characteristic of HUS initiated by Stx in the
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Opinion
TRENDS in Microbiology Vol.9 No.6 June 2001
absence of widespread kidney endothelial cell death17,18? It seems likely that mechanisms other than endothelial apoptosis are important in the initiation of the microvascular thrombosis associated with HUS. The gut and the immune system
Intestinal epithelial cells (IECs) play a pivotal role in the initiation and modulation of the mucosal immune response in the gastrointestinal tract by interacting with immune cells of the lymphoid tissue, lamina propria lymphocytes and intraepithelial lymphocytes19–21. M cells present in the gut epithelium can transport antigens from the lumen to aggregated lymphoid tissues of the Peyer’s patches where macrophages and dendritic cells are located, and can actively participate in antigen degradation and presentation. Under bacterial stimuli, IECs secrete immunomodulatory molecules such as cytokines and chemokines, which alter the local microenvironment within the lamina propria and Peyer’s patches and thereby influence the nature and character of these immune responses. In particular, IEC-derived mediators will affect activation and differentiation of T cells, and the extent to which proinflammatory T-cell subsets are induced. Human gut epithelial cell lines and baboon gut epithelium have been shown to express Gb3 receptors22–24. Therefore, Stx released into the gut during infection with E. coli O157:H7 could further modulate this process either directly or indirectly both in the lamina propria and the Peyer’s patches. In vitro studies have revealed that Stx stimulates IECs to secrete proinflammatory cytokines and neutrophil chemoattractant molecules, such as interleukin (IL)-8 (Ref. 25). It has been shown that Stx translocates across and enters IECs in an energydependent and saturable manner22. Holotoxin uptake is likely to result in the delivery of StxA into the cell cytosol whereas the membrane-bound StxB subunits will undergo anteriograde transport to the basolateral membrane, leading to their delivery to the sub-epithelial layer. Consequently, we favour the view that both StxA and StxB would be able to exert effects on enterocytes readily, but that transcytosed B subunits (now lacking the A subunit) would be able to modulate immune cell function directly in the Peyer’s patches and the lamina propria. Immune activation in HUS
During E. coli O157:H7 gastroenteritis, leukocytosis has been identified as an independent risk factor for the development of HUS (Refs 26,27). To date, the possibility that immune cell activation is involved in the development of HUS has not been explored. Activation of T cells in the gut, which then target the kidney, could explain the mismatch between the clinical features of HUS and the known http://tim.trends.com
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effects of Stx, particularly the delay in the onset of the disease. We have recently demonstrated that StxB directly activates Gb3-expressing colonic epithelial cells (CaCo-2) to induce expression of pro-inflammatory cytokine genes, such as those encoding IL-1α and IL-1β (M. Soriani and T.R. Hirst, unpublished). The ability of Stx to be taken up and to transcytose across the epithelial barrier should deliver the toxin into the lamina propria or Peyer’s patches where it can directly affect leukocyte function28. As discussed earlier, toxin transcytosis is likely to result in the preferential delivery of StxB, rather than intact toxin, to the sub-epithelium, as most of the A subunits will remain in the cytosolic compartment of the epithelial cell29. Thus, it is conceivable that properties associated with StxB are responsible for modulating immune cell activation. In this regard, recent work on a number of AB5 toxins, including cholera toxin, E. coli enterotoxin and Stx, has revealed that interaction between the B subunit components of these toxins and their target cell surface receptors can lead to profound effects on cells of the immune system30–33. Direct interaction of Stx (or its component B subunit) with cells of the immune system, or cytokine release by gut epithelia in response to Stx, might lead to leukocyte activation and migration to the kidney18,34.
‘...Stx-mediated activation of immune cells in the gut initiates the pathology of HUS in the kidney.’ Innate and acquired immune responses to both host- and microorganism-derived stimuli vary considerably between individuals. These responses have been shown to be under complex genetic control and might determine both the susceptibility to and the severity of a variety of infectious conditions35–38. The variability of T-cell responses to specific antigens and superantigens, and the balance between T-helper 1 (Th1) and Th2 CD4+ T cells have been attributed to genetic heterozygosity37,39,40. Therefore, similar innate differences in the T-cell response to Stx or hostderived inflammatory mediators might determine why only 5% of patients with E. coli O157:H7associated gastroenteritis go on to develop HUS. The kidney endothelium as the target in HUS
Why the kidney is the primary target in HUS is uncertain but endothelial cell dysfunction is presumed to be essential for the development of glomerular disease41. Histopathological studies of HUS reveal endothelial swelling, vacuolation and thrombosis of the capillary lumen but not
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TRENDS in Microbiology Vol.9 No.6 June 2001
(b)
Gut
Kidney
(a) HUS
Inflammation
STEC
Intestinal epithelial cells STX StxA StxB
Thrombosis
Neutrophils Lymphocytes
E-selectin ICAM-1 VCAM-1 Gb3 CD40 TF TM Transcytosis
(c) Kidney endothelial cell activation
Inhibition of protein synthesis
Bloodstream transport of activated immune cells and Stx to the kidney
Activation of lymphocytes and other immune cells
Gb3 receptor binding
Apoptosis Cytokines and chemokines Lamina propria Peyer's patches
Stx StxB
TRENDS in Microbiology
Fig. 1. Artwork kindly provided by M. Soriani. The role of immune cell activation in the pathogenesis of post-diarrhoeal haemolytic uraemic syndrome (HUS). (a) Ingestion of Shiga toxin (Stx)-producing Escherichia coli O157 (STEC) leads to release of Stx in the gut. (b) Interaction of Stx with intestinal epithelial cells via Gb3 receptors induces toxin uptake and toxin-mediated inhibition of protein synthesis. StxB interaction with Gb3 might lead to cell apoptosis and/or upregulation of proinflammatory cytokines and chemokines. Some toxin and its component B subunit traverses the epithelial barrier by transcytosis, leading to interaction with, and activation of, immune cells of the lamina propria and/or Peyer’s patches. (c) Activated T cells and other inflammatory cells from the gut migrate via the lymphatic system into the circulation. (d) Upon entry into the glomeruli of the kidney, activated immune cells interact with CD40+ endothelial cells. This leads to upregulation of pro-coagulant factors and adhesion molecules, and elevated expression of Gb3 receptors. The synergistic effect of the immune system and Stx (or StxB) on the endothelium promotes thrombosis, favours neutrophil infiltration and increases susceptibility to Stx, leading eventually to renal failure.
widespread necrosis17,18. In in vitro models, Stx induces endothelial cytotoxicity, which is enhanced by inflammatory mediators such as lipopolysaccharide (LPS), tumour necrosis factor (TNF)-α and IL-1 (Refs 42,43). Highly adapted for filtration, it is also possible that the glomerular endothelium possesses specific cell surface components that trigger the events leading to HUS via a tropism for immune cells activated in the gut and enhanced toxin action34. We suggest that, in the early phases of HUS, T cells activated in the gut orchestrate endothelial dysfunction via a range of soluble and contact-dependent mediators. Recent reports investigating conditions ranging from atherosclerosis to allogeneic transplantation have pointed to the ability of activated T cells to target the vascular endothelium44–46. We propose http://tim.trends.com
that this is an important mechanism through which glomerular function is disrupted. Thrombosis and the endothelium
Normal haemostasis is a complex balance between pro- and anti-thrombotic pathways, which mainly take place on endothelial or platelet cell surfaces47. Endothelial cell death and exposure of basement membrane might explain the renal thrombotic lesions characteristic of HUS. However, in the context of what is known about the biology of other vascular diseases47, endothelial dysfunction rather than death is more likely. We and others have shown that endothelial activation with a variety of inflammatory mediators, including bacterial products (e.g. LPS) and cytokines (e.g. IL-1, TNF-α and IL-8), initiates intravascular thrombosis and enhances the pro-coagulant consequences of endothelial–inflammatory cell interactions48–51. We propose that, in HUS, T-cell engagement of the vessel wall results in glomerular thrombosis52. Recently, it has been shown that interactions between CD40+ endothelial cells and CD154+ on CD4+ T cells upregulate the expression of endothelial adhesion molecules [E-selectin, intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)], immune cell chemoattractants [IL-8, monocyte chemoattractant protein 1 (MCP-1) and RANTES] and the pro-coagulant molecule tissue factor, while downregulating the expression of the anti-coagulant molecule thrombomodulin, thus resulting in a pro-thrombotic surface45,52,53. These molecular
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TRENDS in Microbiology Vol.9 No.6 June 2001
events could be key to the processes of inflammation, neutrophil infiltration and thrombosis, which characterize HUS. Hypothesis
Acknowledgements We would like to thank Professor D.C. Wraith and Dr M. Saleem for their valuable comments during the preparation of this manuscript.
Our hypothesis is that Stx-mediated activation of immune cells in the gut initiates the pathology of HUS in the kidney (Fig. 1). We suggest that this process occurs either alone or in synergy with toxin action on the kidney endothelium. The possibility that Stx has indirect effects on immune cell function via gut epithelial cells is central to this hypothesis. The potential for cytokine production by epithelial cells, leading to the modulation of the microenvironment governing T-cell responsiveness, is well recognized33. Although colonic epithelial cell lines have been shown to produce IL-8 in response to Stx (Refs 25,54), the effect of this and other inflammatory mediators on immune function in the context of HUS has not been previously addressed. Indeed, the hypothesis that T cells are implicated in this process is novel and previously untested. Our hypothesis could be explored in several ways, including in vitro use of gut epithelial cell–T cell co-culture systems. Comparisons between human intestinal cell lines, such as Caco2 cells, which express the AB5 toxin
References 1 Bell, B.P. et al. (1994) A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The Washington experience. J. Am. Med. Assoc. 272, 1349–1353 2 Dundas, S. et al. (1999) Effectiveness of therapeutic plasma exchange in the 1996 Lanarkshire Escherichia coli O157:H7 outbreak. Lancet 354, 1327–1330 3 Taylor, C.M. and Monnens, L.A. (1998) Advances in haemolytic uraemic syndrome. Arch. Dis. Child. 78, 190–193 4 Paton, J.C. and Paton, A.W. (1998) Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11, 450–479 5 Mangeney, M. et al. (1993) Apoptosis induced in Burkitt’s lymphoma cells via Gb3/CD77, a glycolipid antigen. Cancer Res. 53, 5314–5319 6 Jones, N.L. et al. (2000) Escherichia coli Shiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family. Am. J. Physiol. Gastrointest. Liver Physiol. 278, G811–G819 7 Acheson, D.W.K. and Keusch, G.T. (1999) The family of shiga toxins. In A Comprehensive Sourcebook of Bacterial Protein Toxins (Alouf, J.E. and Freer, J.H., eds), pp. 229–242, Academic Press 8 Taguchi, T. et al. (1998) Verotoxins induce apoptosis in human renal tubular epithelium derived cells. Kidney Int. 53, 1681–1688 9 Yoshida, T. et al. (1999) Primary cultures of human endothelial cells are susceptible to low doses of Shiga toxins and undergo apoptosis. J. Infect. Dis. 180, 2048–2052 10 Uchida, H. et al. (1999) Shiga toxins induce apoptosis in pulmonary epithelium-derived cells. J. Infect. Dis. 180, 1902–1911 http://tim.trends.com
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receptor, and T84 cells, which lack detectable Gb3 (Ref. 22), will provide important insights into the mechanisms of these interactions. It will also be important to compare the action of Stx holotoxin with the ‘non-toxic’ StxB pentamer to differentiate between effects arising from interaction of StxB with Gb3 and the inhibition of protein synthesis. Finally, given the difference in the propensity of Stx1 and Stx2 to transcytose the gut epithelium in laboratory models and to cause clinical HUS (Ref. 28), the above comparative experiments should be conducted with these structurally and functionally related proteins. Conclusions
In view of the absence of specific treatments for HUS and the associated mortality, it is important to explore new hypotheses that could herald novel interventions and therapies based on downregulation of inflammatory mediators. Plasmapheresis, which is known to modulate several immune-mediated diseases, has been advocated by some authorities as an adjunctive treatment for HUS (Ref. 2). If our hypothesis is correct, a more targeted antiinflammatory approach aimed at modulating immune cell function could be a promising avenue for further study.
11 Katagiri, Y.U. et al. (1999) Activation of Src family kinase yes induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergent-insoluble microdomains. J. Biol. Chem. 274, 35278–35282 12 Sakiri, R. et al. (1998) Shiga toxin type 1 activates tumor necrosis factor-α gene transcription and nuclear translocation of the transcriptional activators nuclear factor-κB and activator protein-1. Blood 92, 558–566 13 Karmali, M.A. et al. (1985) The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 151, 775–782 14 Tarr, P.I. et al. (1990) Escherichia coli O157:H7 and the hemolytic uremic syndrome: importance of early cultures in establishing the etiology. J. Infect. Dis. 162, 553–556 15 Cornick, N.A. et al. (2000) Shiga toxin-producing Escherichia coli infection: temporal and quantitative relationships among colonization, toxin production, and systemic disease. J. Infect. Dis. 181, 242–251 16 Uzzau, S. and Fasano, A. (2000) Cross-talk between enteric pathogens and the intestine. Cell. Microbiol. 2, 83–89 17 Richardson, S.E. et al. (1988) The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Hum. Pathol. 19, 1102–1108 18 Inward, C.D. et al. (1997) Renal histopathology in fatal cases of diarrhoea-associated haemolytic uraemic syndrome. Pediatr. Nephrol. 11, 556–559 19 Cerf-Bensussan, N. et al. (1984) Intraepithelial lymphocytes modulate Ia expression by intestinal epithelial cells. J. Immunol. 132, 2244–2252
20 Kagnoff, M.F. and Eckmann, L. (1997) Epithelial cells as sensors for microbial infection. J. Clin. Invest. 100, 6–10 21 Hershberg, R.M. et al. (1998) Highly polarized HLA class II antigen processing and presentation by human intestinal epithelial cells. J. Clin. Invest. 102, 792–803 22 Acheson, D.W. et al. (1996) Translocation of Shiga toxin across polarized intestinal cells in tissue culture. Infect. Immun. 64, 3294–3300 23 Taylor, F.B. et al. (1999) Characterization of the baboon responses to Shiga-like toxin: descriptive study of a new primate model of toxic responses to Stx-1. Am. J. Pathol. 154, 1285–1299 24 Jacewicz, M.S. et al. (1995) Maturational regulation of globotriaosylceramide, the Shigalike toxin 1 receptor, in cultured human gut epithelial cells. J. Clin. Invest. 96, 1328–1335 25 Thorpe, C.M. et al. (1999) Shiga toxins stimulate secretion of interleukin-8 from intestinal epithelial cells. Infect. Immun. 67, 5985–5993 26 Fitzpatrick, M.M. et al. (1992) Interleukin-8 and polymorphoneutrophil leukocyte activation in haemolytic uraemic syndrome of childhood. Kidney Int. 42, 951–956 27 Buteau, C. et al. (2000) Leukocytosis in children with Escherichia coli O157:H7 enteritis developing the hemolytic-uremic syndrome. Pediatr. Infect. Dis. J. 19, 642–647 28 Hurley, B.P. et al. (1999) Shiga toxins 1 and 2 translocate differently across polarized intestinal epithelial cells. Infect. Immun. 67, 6670–6677 29 Pelham, H.R. (1992) The secretion of proteins by cells. Proc. R. Soc. London Ser. B Biol. Sci. 250, 1–10
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a genome-wide scan. Proc. Natl. Acad. Sci. U. S. A. 97, 8005–8009 Huleatt, J.W. et al. (2001) Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8 T lymphocytes. J. Immunol. 166, 4065–4073 Papageorgiou, A.C. and Acharya, K.R. (2000) Microbial superantigens: from structure to function. Trends Microbiol. 8, 369–375 Inward, C.D. et al. (1995) Soluble circulating cell adhesion molecules in haemolytic uraemic syndrome. Pediatr. Nephrol. 9, 574–578 van Setten, P.A. et al. (1997) Effects of TNF-α on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells. Kidney Int. 51, 1245–1256 Louise, C.B. et al. (1997) Sensitization of human umbilical vein endothelial cells to Shiga toxin: involvement of protein kinase C and NF-κB. Infect. Immun. 65, 3337–3344 Mach, F. et al. (1998) CD40 signaling in vascular cells: a key role in atherosclerosis? Atherosclerosis 137 (Suppl.), S89–S95 Yellin, M.J. et al. (1995) Functional interactions of T cells with endothelial cells: the role of CD40L–CD40-mediated signals. J. Exp. Med. 182, 1857–1864 Prober, J.S. and Cotran, R. (1990) Cytokines and endothelial cell biology. Physiol. Rev. 70, 427–451 Faust, S.N. et al. (2000) Disseminated intravascular coagulation and purpura fulminans secondary to infection. Baillieres Best Pract. Res. Clin. Haematol. 13, 179–197
48 Heyderman, R.S. et al. (1992) Modulation of the anticoagulant properties of glycosaminoglycans on the surface of the vascular endothelium by endotoxin and neutrophils: evaluation by an amidolytic assay. Thromb. Res. 67, 677–685 49 Heyderman, R.S. et al. (1995) Modulation of the endothelial procoagulant response to lipopolysaccharide and tumour necrosis factor-α in vitro: evaluation of new treatment strategies. Inflamm. Res. 44, 275–280 50 Klein, N.J. et al. (1996) The influence of capsulation and lipooligosaccharide structure on neutrophil adhesion molecule expression and endothelial injury by Neisseria meningitidis. J. Infect. Dis. 173, 172–179 51 Vervloet, M.G. et al. (1998) Derangements of coagulation and fibrinolysis in critically ill patients with sepsis and septic shock. Semin. Thromb. Hemost. 24, 33–44 52 Miller, D.L. et al. (1998) CD40L–CD40 interactions regulate endothelial cell surface tissue factor and thrombomodulin expression. J. Leukocyte Biol. 63, 373–379 53 Thienel, U. et al. (1999) CD154 (CD40L) induces human endothelial cell chemokine production and migration of leukocyte subsets. Cell. Immunol. 198, 87–95 54 Yamasaki, C. et al. (1999) 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. 442, 231–234
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30 Nashar, T.O. et al. (1996) Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD8+ T lymphocytes. Int. Immunol. 8, 731–736 31 Nashar, T.O. et al. (1997) Modulation of B-cell activation by the B subunit of Escherichia coli enterotoxin: receptor interaction up-regulates MHC class II, B7, CD40, CD25 and ICAM-1. Immunology 91, 572–578 32 Williams, N.A. et al. (1997) Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin. Proc. Natl. Acad. Sci. U. S. A. 94, 5290–5295 33 Williams, N.A. et al. (1999) Immune modulation by the cholera-like enterotoxins: from adjuvant to immunotherapeutic. Immunol. Today 20, 95–101 34 te Loo, D.M. et al. (2000) Binding and transfer of verocytotoxin by polymorphonuclear leukocytes in hemolytic uremic syndrome. Blood 95, 3396–3402 35 Nadel, S. et al. (1996) Variation in the tumor necrosis factor-α gene promoter region may be associated with death from meningococcal disease. J. Infect. Dis. 174, 878–880 36 Westendorp, R.G. et al. (1997) Genetic influence on cytokine production and fatal meningococcal disease. Lancet 349, 170–173 37 Reiner, S.L. and Locksley, R.M. (1995) The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13, 151–177 38 Bellamy, R. et al. (2000) Genetic susceptibility to tuberculosis in Africans:
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Opinion
TRENDS in Microbiology Vol.9 No.6 June 2001
Is immune cell activation the missing link in the pathogenesis of postdiarrhoeal HUS? Robert S. Heyderman, Marco Soriani and Timothy R. Hirst Haemolytic uraemic syndrome (HUS), which is caused by Shiga toxin (Stx)producing Escherichia coli, is the commonest cause of acute renal failure in childhood. It is widely believed that HUS develops following the release of Stx, an AB5 toxin that inhibits protein synthesis and has a direct toxic effect on the kidney endothelium. There remains, however, a mismatch between the current understanding of the pathogenesis of HUS and the evolution of the clinical signs of the disease. Our hypothesis is that Stx-mediated immune cell activation in the gut is the missing link in the pathogenesis of this condition, initiating the characteristic renal pathology of HUS either alone or in synergy with Stx. Validation of this hypothesis could lead to a targeted anti-inflammatory approach aimed at modulating immune cell function in HUS.
Robert S. Heyderman* Marco Soriani Timothy R. Hirst Dept of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, UK BS8 1TD. *e-mail: r.heyderman@ bristol.ac.uk
In the past decade, multiple outbreaks of Shiga toxin (Stx)-producing Escherichia coli O157:H7mediated gastroenteritis and haemolytic uraemic syndrome (HUS) have highlighted the global public health impact of this emerging pathogen1–3. HUS occurs in ~5% of E. coli O157:H7-infected individuals with bloody diarrhoea and is characterized by acute renal failure, thrombocytopaenia and microangiopathic haemolytic anaemia. The precise sequence of events leading to the development of HUS is unknown3,4. Although it is widely believed that Stx exerts a direct toxic effect on the endothelium of the kidney, recent evidence suggests that AB5 toxins, particularly the B subunit, have many biological effects and can activate signal transduction pathways, thereby influencing epithelial and leukocyte survival, activation and cell death5,6. These new insights have led us to propose a novel immune-mediated mechanism for the development of E. coli O157:H7-mediated HUS. Stx and pathogenesis
The E. coli Stx family of toxins has two major members: Stx1, which structurally is virtually identical to the Shiga toxin produced by
Shigella dysenteriae type 1, and Stx2, which has only 56% homology to Stx1. All Stx family members have an A subunit (StxA) with RNA-glycohydrolase activity and five B subunits (StxB), which bind with high affinity to the glycolipid receptor Gb3 (CD77) found on the surface of eukaryotic cells7. StxA alone is unable to initiate toxicity because it cannot bind to the Gb3 cell surface receptor. Receptor binding by the holotoxin triggers Stx uptake and trafficking to the endoplasmic reticulum, leading to translocation of the enzymatic A subunit into the cytosol where it catalyses depurination of a single adenine residue in the 28S rRNA of 60S ribosomes, resulting in inhibition of protein synthesis7. Stx also triggers signalling events that lead to apoptosis in endothelial and epithelial cells6,8–10, activation of the Src kinase family11 and nuclear translocation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1)12. Although the contribution of the toxic A subunit to the induction of these events has been demonstrated, it is also clear that the B subunit possesses signalling properties in its own right that can cause apoptosis of cells expressing the Gb3 receptor5,6.
‘...mechanisms other than endothelial apoptosis are important in the initiation of the microvascular thrombosis associated with HUS.’ Limitations of Stx as the sole mediator of HUS
Several features of HUS suggest that Stx-mediated toxicity alone is not sufficient to explain the epidemiological link between Stx and the development of HUS. First, if the glomerular lesions are toxin mediated, why is there a delay of five to seven days between the onset of diarrhoea, when E. coli O157:H7 and its toxin are most easily detected in the stool13,14, and the subsequent development of HUS? It has been suggested that this delay represents the time it takes for Stx to accumulate at its target sites or the time taken for the toxin to induce clinical disease15. However, these explanations fail to account for the poor correlation between the level of intestinal toxin, the clinical outcome of individual patients13 and the abrupt nature of the host inflammatory response associated with HUS. Second, how does Stx, a potent inhibitor of protein synthesis, trigger such a marked inflammatory process with cytokine production and widespread neutrophil activation? Alternative signalling pathways must be involved in the inflammatory response associated with the disease16. Finally, how is the glomerular thrombosis characteristic of HUS initiated by Stx in the
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absence of widespread kidney endothelial cell death17,18? It seems likely that mechanisms other than endothelial apoptosis are important in the initiation of the microvascular thrombosis associated with HUS. The gut and the immune system
Intestinal epithelial cells (IECs) play a pivotal role in the initiation and modulation of the mucosal immune response in the gastrointestinal tract by interacting with immune cells of the lymphoid tissue, lamina propria lymphocytes and intraepithelial lymphocytes19–21. M cells present in the gut epithelium can transport antigens from the lumen to aggregated lymphoid tissues of the Peyer’s patches where macrophages and dendritic cells are located, and can actively participate in antigen degradation and presentation. Under bacterial stimuli, IECs secrete immunomodulatory molecules such as cytokines and chemokines, which alter the local microenvironment within the lamina propria and Peyer’s patches and thereby influence the nature and character of these immune responses. In particular, IEC-derived mediators will affect activation and differentiation of T cells, and the extent to which proinflammatory T-cell subsets are induced. Human gut epithelial cell lines and baboon gut epithelium have been shown to express Gb3 receptors22–24. Therefore, Stx released into the gut during infection with E. coli O157:H7 could further modulate this process either directly or indirectly both in the lamina propria and the Peyer’s patches. In vitro studies have revealed that Stx stimulates IECs to secrete proinflammatory cytokines and neutrophil chemoattractant molecules, such as interleukin (IL)-8 (Ref. 25). It has been shown that Stx translocates across and enters IECs in an energydependent and saturable manner22. Holotoxin uptake is likely to result in the delivery of StxA into the cell cytosol whereas the membrane-bound StxB subunits will undergo anteriograde transport to the basolateral membrane, leading to their delivery to the sub-epithelial layer. Consequently, we favour the view that both StxA and StxB would be able to exert effects on enterocytes readily, but that transcytosed B subunits (now lacking the A subunit) would be able to modulate immune cell function directly in the Peyer’s patches and the lamina propria. Immune activation in HUS
During E. coli O157:H7 gastroenteritis, leukocytosis has been identified as an independent risk factor for the development of HUS (Refs 26,27). To date, the possibility that immune cell activation is involved in the development of HUS has not been explored. Activation of T cells in the gut, which then target the kidney, could explain the mismatch between the clinical features of HUS and the known http://tim.trends.com
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effects of Stx, particularly the delay in the onset of the disease. We have recently demonstrated that StxB directly activates Gb3-expressing colonic epithelial cells (CaCo-2) to induce expression of pro-inflammatory cytokine genes, such as those encoding IL-1α and IL-1β (M. Soriani and T.R. Hirst, unpublished). The ability of Stx to be taken up and to transcytose across the epithelial barrier should deliver the toxin into the lamina propria or Peyer’s patches where it can directly affect leukocyte function28. As discussed earlier, toxin transcytosis is likely to result in the preferential delivery of StxB, rather than intact toxin, to the sub-epithelium, as most of the A subunits will remain in the cytosolic compartment of the epithelial cell29. Thus, it is conceivable that properties associated with StxB are responsible for modulating immune cell activation. In this regard, recent work on a number of AB5 toxins, including cholera toxin, E. coli enterotoxin and Stx, has revealed that interaction between the B subunit components of these toxins and their target cell surface receptors can lead to profound effects on cells of the immune system30–33. Direct interaction of Stx (or its component B subunit) with cells of the immune system, or cytokine release by gut epithelia in response to Stx, might lead to leukocyte activation and migration to the kidney18,34.
‘...Stx-mediated activation of immune cells in the gut initiates the pathology of HUS in the kidney.’ Innate and acquired immune responses to both host- and microorganism-derived stimuli vary considerably between individuals. These responses have been shown to be under complex genetic control and might determine both the susceptibility to and the severity of a variety of infectious conditions35–38. The variability of T-cell responses to specific antigens and superantigens, and the balance between T-helper 1 (Th1) and Th2 CD4+ T cells have been attributed to genetic heterozygosity37,39,40. Therefore, similar innate differences in the T-cell response to Stx or hostderived inflammatory mediators might determine why only 5% of patients with E. coli O157:H7associated gastroenteritis go on to develop HUS. The kidney endothelium as the target in HUS
Why the kidney is the primary target in HUS is uncertain but endothelial cell dysfunction is presumed to be essential for the development of glomerular disease41. Histopathological studies of HUS reveal endothelial swelling, vacuolation and thrombosis of the capillary lumen but not
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Fig. 1. Artwork kindly provided by M. Soriani. The role of immune cell activation in the pathogenesis of post-diarrhoeal haemolytic uraemic syndrome (HUS). (a) Ingestion of Shiga toxin (Stx)-producing Escherichia coli O157 (STEC) leads to release of Stx in the gut. (b) Interaction of Stx with intestinal epithelial cells via Gb3 receptors induces toxin uptake and toxin-mediated inhibition of protein synthesis. StxB interaction with Gb3 might lead to cell apoptosis and/or upregulation of proinflammatory cytokines and chemokines. Some toxin and its component B subunit traverses the epithelial barrier by transcytosis, leading to interaction with, and activation of, immune cells of the lamina propria and/or Peyer’s patches. (c) Activated T cells and other inflammatory cells from the gut migrate via the lymphatic system into the circulation. (d) Upon entry into the glomeruli of the kidney, activated immune cells interact with CD40+ endothelial cells. This leads to upregulation of pro-coagulant factors and adhesion molecules, and elevated expression of Gb3 receptors. The synergistic effect of the immune system and Stx (or StxB) on the endothelium promotes thrombosis, favours neutrophil infiltration and increases susceptibility to Stx, leading eventually to renal failure.
widespread necrosis17,18. In in vitro models, Stx induces endothelial cytotoxicity, which is enhanced by inflammatory mediators such as lipopolysaccharide (LPS), tumour necrosis factor (TNF)-α and IL-1 (Refs 42,43). Highly adapted for filtration, it is also possible that the glomerular endothelium possesses specific cell surface components that trigger the events leading to HUS via a tropism for immune cells activated in the gut and enhanced toxin action34. We suggest that, in the early phases of HUS, T cells activated in the gut orchestrate endothelial dysfunction via a range of soluble and contact-dependent mediators. Recent reports investigating conditions ranging from atherosclerosis to allogeneic transplantation have pointed to the ability of activated T cells to target the vascular endothelium44–46. We propose http://tim.trends.com
that this is an important mechanism through which glomerular function is disrupted. Thrombosis and the endothelium
Normal haemostasis is a complex balance between pro- and anti-thrombotic pathways, which mainly take place on endothelial or platelet cell surfaces47. Endothelial cell death and exposure of basement membrane might explain the renal thrombotic lesions characteristic of HUS. However, in the context of what is known about the biology of other vascular diseases47, endothelial dysfunction rather than death is more likely. We and others have shown that endothelial activation with a variety of inflammatory mediators, including bacterial products (e.g. LPS) and cytokines (e.g. IL-1, TNF-α and IL-8), initiates intravascular thrombosis and enhances the pro-coagulant consequences of endothelial–inflammatory cell interactions48–51. We propose that, in HUS, T-cell engagement of the vessel wall results in glomerular thrombosis52. Recently, it has been shown that interactions between CD40+ endothelial cells and CD154+ on CD4+ T cells upregulate the expression of endothelial adhesion molecules [E-selectin, intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)], immune cell chemoattractants [IL-8, monocyte chemoattractant protein 1 (MCP-1) and RANTES] and the pro-coagulant molecule tissue factor, while downregulating the expression of the anti-coagulant molecule thrombomodulin, thus resulting in a pro-thrombotic surface45,52,53. These molecular
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events could be key to the processes of inflammation, neutrophil infiltration and thrombosis, which characterize HUS. Hypothesis
Acknowledgements We would like to thank Professor D.C. Wraith and Dr M. Saleem for their valuable comments during the preparation of this manuscript.
Our hypothesis is that Stx-mediated activation of immune cells in the gut initiates the pathology of HUS in the kidney (Fig. 1). We suggest that this process occurs either alone or in synergy with toxin action on the kidney endothelium. The possibility that Stx has indirect effects on immune cell function via gut epithelial cells is central to this hypothesis. The potential for cytokine production by epithelial cells, leading to the modulation of the microenvironment governing T-cell responsiveness, is well recognized33. Although colonic epithelial cell lines have been shown to produce IL-8 in response to Stx (Refs 25,54), the effect of this and other inflammatory mediators on immune function in the context of HUS has not been previously addressed. Indeed, the hypothesis that T cells are implicated in this process is novel and previously untested. Our hypothesis could be explored in several ways, including in vitro use of gut epithelial cell–T cell co-culture systems. Comparisons between human intestinal cell lines, such as Caco2 cells, which express the AB5 toxin
References 1 Bell, B.P. et al. (1994) A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The Washington experience. J. Am. Med. Assoc. 272, 1349–1353 2 Dundas, S. et al. (1999) Effectiveness of therapeutic plasma exchange in the 1996 Lanarkshire Escherichia coli O157:H7 outbreak. Lancet 354, 1327–1330 3 Taylor, C.M. and Monnens, L.A. (1998) Advances in haemolytic uraemic syndrome. Arch. Dis. Child. 78, 190–193 4 Paton, J.C. and Paton, A.W. (1998) Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11, 450–479 5 Mangeney, M. et al. (1993) Apoptosis induced in Burkitt’s lymphoma cells via Gb3/CD77, a glycolipid antigen. Cancer Res. 53, 5314–5319 6 Jones, N.L. et al. (2000) Escherichia coli Shiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family. Am. J. Physiol. Gastrointest. Liver Physiol. 278, G811–G819 7 Acheson, D.W.K. and Keusch, G.T. (1999) The family of shiga toxins. In A Comprehensive Sourcebook of Bacterial Protein Toxins (Alouf, J.E. and Freer, J.H., eds), pp. 229–242, Academic Press 8 Taguchi, T. et al. (1998) Verotoxins induce apoptosis in human renal tubular epithelium derived cells. Kidney Int. 53, 1681–1688 9 Yoshida, T. et al. (1999) Primary cultures of human endothelial cells are susceptible to low doses of Shiga toxins and undergo apoptosis. J. Infect. Dis. 180, 2048–2052 10 Uchida, H. et al. (1999) Shiga toxins induce apoptosis in pulmonary epithelium-derived cells. J. Infect. Dis. 180, 1902–1911 http://tim.trends.com
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receptor, and T84 cells, which lack detectable Gb3 (Ref. 22), will provide important insights into the mechanisms of these interactions. It will also be important to compare the action of Stx holotoxin with the ‘non-toxic’ StxB pentamer to differentiate between effects arising from interaction of StxB with Gb3 and the inhibition of protein synthesis. Finally, given the difference in the propensity of Stx1 and Stx2 to transcytose the gut epithelium in laboratory models and to cause clinical HUS (Ref. 28), the above comparative experiments should be conducted with these structurally and functionally related proteins. Conclusions
In view of the absence of specific treatments for HUS and the associated mortality, it is important to explore new hypotheses that could herald novel interventions and therapies based on downregulation of inflammatory mediators. Plasmapheresis, which is known to modulate several immune-mediated diseases, has been advocated by some authorities as an adjunctive treatment for HUS (Ref. 2). If our hypothesis is correct, a more targeted antiinflammatory approach aimed at modulating immune cell function could be a promising avenue for further study.
11 Katagiri, Y.U. et al. (1999) Activation of Src family kinase yes induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergent-insoluble microdomains. J. Biol. Chem. 274, 35278–35282 12 Sakiri, R. et al. (1998) Shiga toxin type 1 activates tumor necrosis factor-α gene transcription and nuclear translocation of the transcriptional activators nuclear factor-κB and activator protein-1. Blood 92, 558–566 13 Karmali, M.A. et al. (1985) The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 151, 775–782 14 Tarr, P.I. et al. (1990) Escherichia coli O157:H7 and the hemolytic uremic syndrome: importance of early cultures in establishing the etiology. J. Infect. Dis. 162, 553–556 15 Cornick, N.A. et al. (2000) Shiga toxin-producing Escherichia coli infection: temporal and quantitative relationships among colonization, toxin production, and systemic disease. J. Infect. Dis. 181, 242–251 16 Uzzau, S. and Fasano, A. (2000) Cross-talk between enteric pathogens and the intestine. Cell. Microbiol. 2, 83–89 17 Richardson, S.E. et al. (1988) The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Hum. Pathol. 19, 1102–1108 18 Inward, C.D. et al. (1997) Renal histopathology in fatal cases of diarrhoea-associated haemolytic uraemic syndrome. Pediatr. Nephrol. 11, 556–559 19 Cerf-Bensussan, N. et al. (1984) Intraepithelial lymphocytes modulate Ia expression by intestinal epithelial cells. J. Immunol. 132, 2244–2252
20 Kagnoff, M.F. and Eckmann, L. (1997) Epithelial cells as sensors for microbial infection. J. Clin. Invest. 100, 6–10 21 Hershberg, R.M. et al. (1998) Highly polarized HLA class II antigen processing and presentation by human intestinal epithelial cells. J. Clin. Invest. 102, 792–803 22 Acheson, D.W. et al. (1996) Translocation of Shiga toxin across polarized intestinal cells in tissue culture. Infect. Immun. 64, 3294–3300 23 Taylor, F.B. et al. (1999) Characterization of the baboon responses to Shiga-like toxin: descriptive study of a new primate model of toxic responses to Stx-1. Am. J. Pathol. 154, 1285–1299 24 Jacewicz, M.S. et al. (1995) Maturational regulation of globotriaosylceramide, the Shigalike toxin 1 receptor, in cultured human gut epithelial cells. J. Clin. Invest. 96, 1328–1335 25 Thorpe, C.M. et al. (1999) Shiga toxins stimulate secretion of interleukin-8 from intestinal epithelial cells. Infect. Immun. 67, 5985–5993 26 Fitzpatrick, M.M. et al. (1992) Interleukin-8 and polymorphoneutrophil leukocyte activation in haemolytic uraemic syndrome of childhood. Kidney Int. 42, 951–956 27 Buteau, C. et al. (2000) Leukocytosis in children with Escherichia coli O157:H7 enteritis developing the hemolytic-uremic syndrome. Pediatr. Infect. Dis. J. 19, 642–647 28 Hurley, B.P. et al. (1999) Shiga toxins 1 and 2 translocate differently across polarized intestinal epithelial cells. Infect. Immun. 67, 6670–6677 29 Pelham, H.R. (1992) The secretion of proteins by cells. Proc. R. Soc. London Ser. B Biol. Sci. 250, 1–10
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a genome-wide scan. Proc. Natl. Acad. Sci. U. S. A. 97, 8005–8009 Huleatt, J.W. et al. (2001) Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8 T lymphocytes. J. Immunol. 166, 4065–4073 Papageorgiou, A.C. and Acharya, K.R. (2000) Microbial superantigens: from structure to function. Trends Microbiol. 8, 369–375 Inward, C.D. et al. (1995) Soluble circulating cell adhesion molecules in haemolytic uraemic syndrome. Pediatr. Nephrol. 9, 574–578 van Setten, P.A. et al. (1997) Effects of TNF-α on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells. Kidney Int. 51, 1245–1256 Louise, C.B. et al. (1997) Sensitization of human umbilical vein endothelial cells to Shiga toxin: involvement of protein kinase C and NF-κB. Infect. Immun. 65, 3337–3344 Mach, F. et al. (1998) CD40 signaling in vascular cells: a key role in atherosclerosis? Atherosclerosis 137 (Suppl.), S89–S95 Yellin, M.J. et al. (1995) Functional interactions of T cells with endothelial cells: the role of CD40L–CD40-mediated signals. J. Exp. Med. 182, 1857–1864 Prober, J.S. and Cotran, R. (1990) Cytokines and endothelial cell biology. Physiol. Rev. 70, 427–451 Faust, S.N. et al. (2000) Disseminated intravascular coagulation and purpura fulminans secondary to infection. Baillieres Best Pract. Res. Clin. Haematol. 13, 179–197
48 Heyderman, R.S. et al. (1992) Modulation of the anticoagulant properties of glycosaminoglycans on the surface of the vascular endothelium by endotoxin and neutrophils: evaluation by an amidolytic assay. Thromb. Res. 67, 677–685 49 Heyderman, R.S. et al. (1995) Modulation of the endothelial procoagulant response to lipopolysaccharide and tumour necrosis factor-α in vitro: evaluation of new treatment strategies. Inflamm. Res. 44, 275–280 50 Klein, N.J. et al. (1996) The influence of capsulation and lipooligosaccharide structure on neutrophil adhesion molecule expression and endothelial injury by Neisseria meningitidis. J. Infect. Dis. 173, 172–179 51 Vervloet, M.G. et al. (1998) Derangements of coagulation and fibrinolysis in critically ill patients with sepsis and septic shock. Semin. Thromb. Hemost. 24, 33–44 52 Miller, D.L. et al. (1998) CD40L–CD40 interactions regulate endothelial cell surface tissue factor and thrombomodulin expression. J. Leukocyte Biol. 63, 373–379 53 Thienel, U. et al. (1999) CD154 (CD40L) induces human endothelial cell chemokine production and migration of leukocyte subsets. Cell. Immunol. 198, 87–95 54 Yamasaki, C. et al. (1999) 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. 442, 231–234
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30 Nashar, T.O. et al. (1996) Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD8+ T lymphocytes. Int. Immunol. 8, 731–736 31 Nashar, T.O. et al. (1997) Modulation of B-cell activation by the B subunit of Escherichia coli enterotoxin: receptor interaction up-regulates MHC class II, B7, CD40, CD25 and ICAM-1. Immunology 91, 572–578 32 Williams, N.A. et al. (1997) Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin. Proc. Natl. Acad. Sci. U. S. A. 94, 5290–5295 33 Williams, N.A. et al. (1999) Immune modulation by the cholera-like enterotoxins: from adjuvant to immunotherapeutic. Immunol. Today 20, 95–101 34 te Loo, D.M. et al. (2000) Binding and transfer of verocytotoxin by polymorphonuclear leukocytes in hemolytic uremic syndrome. Blood 95, 3396–3402 35 Nadel, S. et al. (1996) Variation in the tumor necrosis factor-α gene promoter region may be associated with death from meningococcal disease. J. Infect. Dis. 174, 878–880 36 Westendorp, R.G. et al. (1997) Genetic influence on cytokine production and fatal meningococcal disease. Lancet 349, 170–173 37 Reiner, S.L. and Locksley, R.M. (1995) The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13, 151–177 38 Bellamy, R. et al. (2000) Genetic susceptibility to tuberculosis in Africans:
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