Enders, G. A.

Hatz, R. A.

English

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Surgical Research – General SubjectsEur Surg Res 2002;34:18–21Surgical Research and Helicobacterpylori Infection – A Contradiction?G.A. Endersa R.A. HatzbaInstitute for Surgical Research and bDepartment of Surgery, LMU Munich, Klinikum Grosshadern,Munich, GermanyG.A. Enders, MDInstitute for Surgical Research, LMU MünchenMarchioninistrasse 27, D–81366 München (Germany)Tel. +49 89 7095 4406, Fax +49 89 7095 8897E-Mail enders@icf.med.uni-muenchen.deABCFax + 41 61 306 12 34E-Mail karger@karger.chwww.karger.com© 2002 S. Karger AG, Basel0014–312X/02/0342–0018$18.50/0Accessible online at:www.karger.com/journals/esrKey WordsHelicobacter pylori W Gastritis W Peyer’s patches WELAM-1 W ICAM-1 W TNF-·IntroductionHistorically surgical research has dealt mostly withtechniques to improve surgery on e.g. operation on theopen heart, organ transplantation, minimal invasive sur-gery and peri- and postoperative pathophysiology. In thisregard there were fruitful research activities in the field ofsepsis, microcirculation, neurotrauma or the develop-ment of lithotripsy of kidney and gallstones [1–6]. Thisresearch has mostly been performed in model systemsincluding animal experiments.The availability of new techniques in molecular biolo-gy has boosted the research in a way that now samplesroutinely taken from patients for diagnositic purposes canbe used to be analysed by these methods and bring furtherinsights into the pathology/pathophysiology of the dis-eases. This can be illustrated by the analysis of relevantexpressed genes within the course of H. pylori related gas-tritis where data from basic immunology of the GI-tractcan be illustrated in a relevant human disease.The understanding of the regulation of an immuneresponse in the gut is of interest for many different dis-eases. The hyperreactivity to food components like in coe-liac disease and food allergy or the autoimmune reactionin chronic inflammatory bowel disease may illustrate this.On the other hand the induction of oral tolerance or theunderstanding why bacteria can not only colonize theintestine but also can induce severe infections and eventu-ally transmigrate and end in a septic syndrome in e.g.immunocompromized patients are all interesting ques-tions which could be answered by a better understandingof the mucosal immune system. In the investigation of GIimmunology surgical models gave new insights into thefunction of Peyer’patches for the organisation of the localimmune system in the intestine. Using traditional tech-niques of removing the patches it became clear that effec-tor cells in the Peyer’s patches like M-cells (fig. 1) areimportant players for initializing the immune response.After reaching contact with luminally presented antigens,they transfer this information to immune cells in thepatches which migrate to the mesenteric lymph nodeswhere the local immune response becomes organized end-ing with the spread of effector cells into the lamina pro-pria and the intraepithelial space of the small intestine[7–9]. This theory is now well appreciated and finds fur-ther support with the notion that the transfer of the prionprotein form BSE might find its way by this pathway [10].The organisation of the immune system of the stomach onthe other hand is not as well defined. There are usually nolymph follicles resembling the Peyer’s patches and bymorphology no antigen presenting cells like M-cells in theepithelial layer despite reports on the function of epithe-Surgical Research and H. pylori Infection Eur Surg Res 2002;34:18–21 19Fig. 1. Scanning electron microscopy of the epithelium covering aPeyer’ patch of the rat. Two M-cells are clearly visible in the middlecharacterized by the microfolds (!3,600).Fig. 2. Analysis of IL-8 expression in biopsy specimen by RT-PCR.Lane 1–4 are from controls, lanes 5–9 from H. pylori associated gas-tritis. H = Stimulated HUVEC as positive control, M = molecularweight marker. Fragment size on a 1% agarose gel is 255 bp. Amplifi-cation was for 35 cycles.lial cells as antigen presenting cells in other parts of theintestine [11–13]. There are early reports on the inductionof an immune response in the stomach resulting in a hor-mone response as well as in hypersensitivity [14–16]. Amore challenging view on the organization of the immunesystem in the stomach came with the observation that abacterium is the causative agent in the induction of type Bgastritis, ulcer disease and eventually cancer. Helicobacterpylori has been recognized to be responsible for these dis-ease [17, 18]. So the question arises on the colonizationand the regulation of the local immune response with spe-cial reference to how a protective immune response mightbe inducible.There is little known about how H. pylori initially over-comes host defence and persists in a hostile environment.The fact that many patients who harbour the bacteriumremain asymptomatic without developing manifest dis-ease made it a model for investigating the time course andseverity of the immune response in the stomach as well asfor studying strain differences of the bacterium. The pow-er of molecular biologic methods made it possible to gaininsight on sequential gene expression by means of analys-ing the amount of specific RNA expressed in biopsies tak-en from patients by e.g. RT-PCR. One or two biopsy spec-imens are usually enough for the analysis of up to 10 dif-ferent genes including standardization for a housekeepinggene. Using such methods genes activated during thedevelopment of an inflammatory response as well asimmune regulation can be easily analysed during thecourse of a disease.Materials and MethodsBiopsies were taken from patients during diagnostic endoscopy.Usually the equivalent of one biopsy was used to isolate total RNA.After homogenization of the biopsy in lysis puffer the total RNA wasisolated by its selective binding to a silica-matrix in spin columns(RNAeasy; Qiagen, Hilden, Germany). After elution RNA was pre-cipitated and either stored at –70°C or used for reverse transcrip-tion. The cDNA was prepared according to a standard protocol usingMMLV-reverse transcriptase (Gibco BRL; Paisley, Scotland).The amplification of a particular target gene by PCR was per-formed as described [19]. Specific primers were selected from knownsequences and extensively tested against a data bank (GenBank,BLAST). PCR was quantified after different cycle numbers on ethid-ium bromide stained agarose gels using a densitometer with a sophis-ticated software and normalized for housekeeping genes.Results and DiscussionUsing this method it became clear that in the earlyphase of the infection the contact of the bacterium with theepithelial layer of the stomach induces severe chemokineproduction which is able to attract granulocytes into the20 Eur Surg Res 2002;34:18–21 Enders/HatzFig. 3. ICAM-1 (CD54) expression on control (lanes 1–5 ) and H.pylori associated gastritis (lanes 6–11). H = Stimulated HUVEC aspositive control, M = molecular weight marker. Fragment size is238 bp. Amplification was for 30 cycles.Fig. 4. TNF-· expression in biopsy specimen from control (lanes 1–4) and H. pylori associated gastritis (lanes 5–9). M is the molecularweight marker, C are stimulated macrophages as positive control.Fragment size is 444 bp, amplification for 35 cycles.Fig. 5. IL-1ß expression in biopsy specimen from control (lanes 1–4)and H. pylori associated gastritis (lanes 5–9). M is the molecularweight marker, C are LPS stimulated PBL as positive control. Frag-ment size is 447 bp, amplification for 35 cycles.site of inflammation. As shown in figure 2 abundant IL8message could be detected in the biopsy of patients withgastritis. So the presence of the bacterium in the stomachseems to be a trigger for local chemokine expression. Thisobservation could also be extended to the more epithelialcell specific chemokine ENA-78 as shown previously [20].Attraction and activation of granulocytes can be inducedby these cytokines. This induction was dependent on theadherence of the bacterium to epithelial cells as shown byin vitro stimulation experiments using gastric cell lines[21]. Now chemokines are able to attract granulocytes aswell as lymphocytes. For granulocytes to leave the vessel itis important that surface receptors on the granulocytesinteract with adhesion molecules on the local endothelialcells. This interaction has been shown to either inducerolling of cells along the vessel wall or stick before emi-grating out of the blood vessel. The former is mediated bythe interaction of CD62P on the vessel and its ligandPSGL-1 or CD62P on the blood cells. Solid adherence onthe other hand is mediated by the interaction of integrinson blood cells and CD54 ( ICAM-1) on the endothelialwall. ICAM 1 (fig. 3) was found expressed in all biopsieswith H. pylori associated gastritis and controls. The ex-pression could also be shown by immunohistochemistrywhere higher expression was found in gastritis. As counterreceptor on migrating cells the ß-2 integrin CD11/18 hete-rodimer is effective. In this regard we and others were ableto show that soluble H. pylori products had the ability toinduce the upregulation of CD11b on granulocytes [22].Inducible adhesion molecules might be more effectivein the attraction of activated blood cells. Candidates areCD62E and CD 106 (VCAM) as well as the early markerCD62P are candidates. With the exception of CD62E allof these molecules could be found upregulated on endo-thelial cells in gastritis specimens [19]. Apart from theother molecules studied VCAM was not only detected onsome endothelial cells but also on follicular dendritic cellsin lymph follicles which appear in the lamina propriaafter H. pylori infection [23]. As CD62 E (ELAM-1) isfound in chronic inflammatory lesions of the bowel it wassurprising that it could not be detected in H. pylori associ-ated gastritis since potent inducers for CD62E like IL1-ßand TNF-· were detected in the same biopsies. Interest-ingly, whereas TNF-· was induced (fig. 4) IL1-ß was con-stitutively expressed in all samples (fig. 5). IL1-ß has beenshown to have dramatic effects on the acid output of thestomach [24, 25]. This seems to suggest that acid produc-tion could be regulated by a different pathway in healthand disease. On the other hand TNF-· is a potent inducerof adhesion molecules on endothelial cells. So it may wellbe that in the stomach adhesion molecule regulation dif-fers from other parts of the intestine.With the help of molecular biological methods it is nowpossible to get data from routine biopsies which expandthe knowledge on the pathogenesis of a particular disease.In this respect H. pylori induced gastritis serves as a para-digm for an immune response in the stomach. Not onlycan the local reaction of the patient be analysed and fol-lowed after eradication but also reasons for recurrence ofthe disease studied. In addition basic data on the immuneregulation in the stomach can be obtained. However, whypatients who are Helicobacter pylori infected vary in theirclinical presentation of manifest disease from beingSurgical Research and H. pylori Infection Eur Surg Res 2002;34:18–21 21asymptomatic to developing gastric carcinoma remains abaffling question. A broad variation in the genetic reper-toire of the patient as well as of the bacterium may be theanswer. With the disclosure of the complete H. pylorigenome every variation and gene shift should be detect-able [26]. Even the publication of the human genomeoffers additional chances to evaluate differences in thereaction of the immune system in the stomach wall.So is this the end of H. pylori research in the stomachwith the description of the pathogenesis and the possibili-ty to eradicate the bug? Eradication studies have shownthat some changes in the pathology persist even after lon-ger periods of time [27]. For example a possible increasein Barrett carcinomas after eradication has been observed[28–30]. So H. pylori infection may even have some bene-ficial effects on some unknown defence mechanism as forexample acting protective on the gastro-oesophageal junc-tion. These new aspects are open for investigations on thegenetic repertoire expressed by the bacterium as well as bythe particular patient. So the combination of a surgeonhaving access to human tissues and a powerful lab usingmolecular biological methods allows the investigation ofthe pathophysiological events and offers new insights intoclinically relevant disease.AcknowledgementThis work has been supported by grants from the LMU (WeigandStiftung) and the Chiles foundation, Portland, Oregon.References1 Messmer K: Basic surgical research. Langen-becks Arch Surg 1998;383:297–299.2 Messmer K, Krombach F: Microcirculation re-search in experimental surgery. Chirurg 1998;69:333–338.3 Baethmann A, Kempski OS: Pathophysiologyof brain edema. Zentralbl Neurochir 1997;58:20–29.4 Brendel W: Shock wave lithotripsy of the kid-ney and gallbladder. Verh Dtsch Ges Inn Med1986;92:168–174.5 Delius M, Brendel W: Historical roots of litho-tripsy. J Lithotr Stone Dis 1990;2:161–163.6 Brendel W, Enders G: Shock waves for gall-stones: Animal studies. Lancet 1983;i:1054.7 Enders GA, Ballhaus S, Brendel W: The in-fluence of Peyer’s patches on the organ-specificdistribution of IgA plasma cells. Immunology1988;63:411–414.8 Enders GA, Delius M, Ballhaus S, Brendel W:Role of Peyer’s patch in the intestinal immuneresponse to cholera toxin in enterically immu-nized rats. Infect Immun 1987;55:1997–1999.9 Enders G, Gottwald T, Brendel W: Inductionof oral tolerance in rats without Peyer’spatches. Immunology 1986;58:311–314.10 Heppner FL, Christ AD, Klein MA, Prinz M,Fried M, Kraehenbuhl JP, Aguzzi A: Transepi-thelial prion transport by M cells. Nat Med2001;7:976–977.11 Bland PW, Warren LG: Antigen presentationby epithelial cells of the rat small intestine. I.Kinetics, antigen specificity and blocking byanti-Ia antisera. Immunology 1986;58:1–7.12 Mayer L, Shlien R: Evidence for function of Iamolecules on gut epithelial cells in man. 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Lancet 1984;i:1311–1315.18 Blaser MJ: Hypotheses on the pathogenesis andnatural history of Helicobacter pylori-inducedinflammation. Gastroenterology 1992;102:720–727.19 Hatz RA, Rieder G, Stolte M, Bayerdorffer E,Meimarakis G, Schildberg FW, Enders G: Pat-tern of adhesion molecule expression on vascu-lar endothelium in Helicobacter pylori-associ-ated antral gastritis. Gastroenterology 1997;112:1908–1919.20 Rieder G, Einsiedl W, Hatz RA, Stolte M,Enders GA, Walz A: Comparison of CXC che-mokines ENA-78 and interleukin-8 expressionin Helicobacter pylori-associated gastritis. In-fect Immun 2001;69:81–88.21 Rieder G, Hatz RA, Moran AP, Walz A, StolteM, Enders G: Role of adherence in interleukin-8 induction in Helicobacter pylori-associatedgastritis. 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Aguzzi A: Transepithelial prion transport by M cells.

Basic surgical research. Langenbecks Arch Surg

Enders G: Characterization of lymphocytic infiltrates in Helicobacter pylori-associated gastritis.

Enders G: Role of adherence

Enders G: Shock waves for gallstones: Animal studies. Lancet

Evidence for function of Ia molecules on gut epithelial cells in man.

Hatz R: Expression of adhesion molecules on human granulocytes after stimulation with Helicobacter pylori membrane proteins: Comparison with membrane proteins from other bacteria. Infect Immun

Hatz RA: Inducible nitric oxide synthase expression before and after eradication of Helicobacter pylori in different forms of gastritis.

Helicobacter pylori genetic diversity and risk of human disease.

Historical roots of lithotripsy.

Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation. Gastroenterology 1992;102:

induction in Helicobacter pylori-associated gastritis. Infect Immun

Induction of oral tolerance in rats without Peyer’s patches. Immunology

Interleukin-1 inhibits the secretion of gastric acid in rats: Possible involvement of prostaglandin.

JE: CagA-positive strains of Helicobacter pylori may protect against Barrett’s esophagus.

Kempski OS: Pathophysiology of brain edema. Zentralbl Neurochir 1997;58:

Krombach F: Microcirculation research in experimental surgery. Chirurg

L: The role of epithelial cells in immune regulation in the gut. Semin Immunol

LG: Antigen presentation by epithelial cells of the rat small intestine. I. Kinetics, antigen specificity and blocking by anti-Ia antisera.

Linking Helicobacter pylori to gastric cancer.

Mechanisms underlying the protective effects of interleukin 1 in experimental nonsteroidal anti-inflammatory drug gastropathy.

RD: The complete genome sequence of the gastric pathogen Helicobacter pylori.

RE: Divalent hapten-induced intestinal anaphylaxis in the mouse enhances macromolecular uptake from the stomach. Gastroenterology

Role of Peyer’s patch in the intestinal immune response to cholera toxin in enterically immunized rats. Infect Immun 1987;55:1997–1999.

Schildberg FW, Enders G: Pattern of adhesion molecule expression on vascular endothelium in Helicobacter pylori-associated antral gastritis. Gastroenterology

Schildberg FW: Mediators of antigen-induced gastrin release: Role of antigen-antibody complexes and the complement system. Res Exp Med (Berl)

Shock wave lithotripsy of the kidney and gallbladder. Verh Dtsch Ges Inn Med

Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet

W: Antigeninduced gastrin release: An immunologic mechanism of gastric antral mucosa. Adv Exp Med Biol

W: The influence of Peyer’s patches on the organ-specific distribution of IgA plasma cells. Immunology

Surgical research and Helicobacter pylori infection - A contradiction?

Universität München: Elektronischen Publikationen

G.A. Enders

R.A. Hatz

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Surgical Research and <i>Helicobacter pylori</i> Infection – A Contradiction?

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Surgical research and Helicobacter pylori infection - A contradiction?

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