Virchows Arch (2009) 455:225–233 DOI 10.1007/s00428-009-0825-8
ORIGINAL ARTICLE
Neutrophil gelatinase-associated lipocalin (NGAL/Lcn2) is upregulated in gastric mucosa infected with Helicobacter pylori Warner Alpízar-Alpízar & Ole Didrik Laerum & Martin Illemann & José A. Ramírez & Adriana Arias & Wendy Malespín-Bendaña & Vanessa Ramírez & Leif R. Lund & Niels Borregaard & Boye Schnack Nielsen
Received: 22 June 2009 / Revised: 13 August 2009 / Accepted: 14 August 2009 / Published online: 1 September 2009 # Springer-Verlag 2009
Abstract Helicobacter pylori infection is one of the most significant risk factors for gastric cancer. The infection is established early in life and persists lifelong leading to a sustained chronic inflammation. Iron is essential for most living organisms. Bacteria use several mechanisms to acquire iron from their hosts, including the synthesis of the potent iron chelators known as siderophores. Hosts cells may express the siderophore-binding protein neutrophil gelatinase-associated lipocalin (NGAL/lipocalin-2 (Lcn2)) in response to infection, thus preventing bacterial iron uptake. We have characterized here the pattern of expression of NGAL/Lcn2 in gastric mucosa (45 non-neoplastic and 38 neoplastic tissue samples) and explored the connection between NGAL/Lcn2 expression and H. pylori
infection. Immunohistochemical analysis showed high NGAL/Lcn2 expression in normal and gastritis-affected mucosa compared to low expression in intestinal metaplasia, dysplasia, and gastric cancer. In normal and gastritisaffected mucosa (n=36 tissue samples), NGAL/Lcn2 was more frequently seen in epithelial cells located at the neck and base of the glands in H. pylori-positive cases than in similar epithelial cells of noninfected cases (Fisher’s exact test, p=0.04). In conclusion, the high expression of NGAL/ Lcn2 in normal and gastritis-affected mucosa infected with H. pylori suggests that NGAL/Lcn2 is upregulated locally in response to this bacterial infection. It is discussed whether this may have a causal relation to the development of gastric cancer.
W. Alpízar-Alpízar : O. D. Laerum The Gade Institute, University of Bergen and Department of Pathology, Haukeland University Hospital, Bergen, Norway
L. R. Lund Department of Biology, Section for Cell and Developmental Biology, University of Copenhagen, Copenhagen, Denmark
W. Alpízar-Alpízar : W. Malespín-Bendaña : V. Ramírez Cancer Research Program, Health Research Institute (INISA), University of Costa Rica, San José, Costa Rica
N. Borregaard The Granulocyte Research Laboratory, Department of Haematology, University of Copenhagen, Rigshospitalet, Copenhagen, Denmark
W. Alpízar-Alpízar : M. Illemann : B. S. Nielsen The Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
W. Alpízar-Alpízar (*) The Gade Institute, Department of Pathology, Haukeland University Hospital, 5021 Bergen, Norway e-mail: awarnercr@yahoo.com e-mail: Warner.Alpizar@student.uib.no
J. A. Ramírez : A. Arias Department of Pathology, Dr. Rafael A. Calderón Guardia Hospital, San José, Costa Rica
Present Address: B. S. Nielsen Exiqon A/S, Diagnostic Product Development, Vedbæk, Denmark
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Keywords NGAL/Lcn2 . Helicobacter pylori . Inflammation . Gastritis . Intestinal metaplasia . Gastric cancer Abbreviations IM Intestinal metaplasia mAb Monoclonal antibody pAbs Polyclonal antibodies TNP Trinitrophenyl hapten NGAL Neutrophil gelatinase-associated lipocalin Lcn2 Lipocalin-2
Introduction Gastric cancer is the second most common cause of cancer deaths worldwide [1]. It is the final result of a multistep process initiated by environmental factors, including diet and Helicobacter pylori infection [2, 3]. H. pylori infection is one of the most important risk factors for this malignancy [4, 5]. The infection is usually established early in life and persists lifelong in the absence of treatment [6]. The infection leads to a sustained chronic inflammation characterized by infiltration of a number of inflammatory cells in the gastric mucosa and expression of inflammatory mediators by immune and epithelial cells [7]. It is the combination of bacterial factors, host immune response, and environmental insults that drives the stepwise transformation starting with mucosal atrophy through metaplasia and dysplasia to overt gastric cancer [8, 9]. Iron is an essential element for a number of metabolic processes in almost all living organisms. During infection, bacteria utilize several mechanisms to obtain iron from their hosts, including synthesis of so-called siderophores, which chelate Fe3+ with high affinity and facilitate its transport into the pathogen [10–12]. An important host defense mechanism, which interferes with bacterial uptake of iron has been unraveled by the discovery of neutrophil gelatinaseassociated lipocalin (NGAL or lipocalin-2 (Lcn2)) [13, 14] or NGAL-homologous proteins (24p3 in mouse and NRL in rats) [15] as siderophore-binding proteins [16]. NGAL/Lcn2 binds some bacterial siderophores and prevents their uptake into bacteria [17]. NGAL/Lcn2 expression has been shown to be upregulated in response to inflammation in epithelial cells of several mucosal surfaces including the gastrointestinal tract and the lower respiratory tract [18–20]. Elevated expression of the NGAL-homologous proteins has also been found in animal models (mice and rats) in response to bacterial infections [21, 22]. The significance of this was demonstrated in a mouse model, where NGAL-deficient mice challenged with a clinical strain of Escherichia coli (H9049 strain) had a substantial increase in bacteremia and
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bacterial burden in some organs, including liver and spleen and a strikingly decreased survival compared to the wildtype mice [23], and also in response to pulmonary Klebsiella pneumoniae infection [22]. Iron is fundamental for the pathogenesis of H. pylori. The ability of H. pylori to acquire iron is considered important for colonization, persistence, and virulence of this microorganism in the gastric mucosa [24–27]. Expression of NGAL/Lcn2 in the stomach has been reported both at the protein and mRNA level [14, 28]. In a mouse model, immunoreactivity for the NGAL-homologous protein, 24p3, was observed in epithelial, endothelial, and infiltrating inflammatory cells in the stomach in response to various forms of gastrointestinal injury [29]. Recently, increased expression of NGAL/Lcn2 was reported in rhesus macaques challenged with H. pylori [30]. These observations suggest that NGAL/Lcn2 is expressed in gastric mucosa in response to inflammation/infection. Here, we study the expression of NGAL/Lcn2 in human gastric mucosa and show for the first time that NGAL/Lcn2 is upregulated in response to H. pylori infection and inflammation.
Material and methods Tissue samples Formalin-fixed and paraffin-embedded tissue samples were obtained from patients undergoing surgery for gastric cancer in two hospitals in Costa Rica (Max Peralta Hospital and Rafael Angel Calderón Guardia Hospital) and one in Bergen, Norway (Haukeland University Hospital). The samples encompassed non-neoplastic mucosa adjacent to the malignant tissue (n=45; 37 from Costa Rica and eight from Norway) and neoplastic lesions (n=38; 32 from Costa Rica and six from Norway). Three to four micrometer thick paraffin-embedded tissue sections were cut from the nonneoplastic material, stained with H&E, and evaluated by a pathologist (ODL). Among the 45 non-neoplastic sections, there were foci characterized as normal mucosa (with some degree of inflammation; n=6), gastritis (n=30), intestinal metaplasia (IM; n=17), and dysplasia (n=11). Information regarding the histopathological subtype of gastric cancer was collected for all 38 neoplastic lesions. The histopathological classification was according to Laurén’s classification system (Norwegian cases) and Japanese classification system (Costa Rican cases). For the purposes of this study, Costa Rican cases were reclassified according to Laurén’s classification system following established criteria given by the Japanese Gastric Cancer Association [31]. Of the 38 cases, 20 were classified as intestinal subtype and 18 as diffuse subtype, and they were all non-cardia cancers (from corpus and antrum regions of the stomach). The study was
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approved by the ethics committees and institutional review boards of each institution (Costa Rica: VI 742-94-571, VI 742-99-340; Norway: REK 053228) and performed in accordance with the World Medical Association Declaration of Helsinki, 1996.
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Negative controls The sections were pretreated in the same way as described above for the NGAL antibody. The mouse NGAL monoclonal antibody was substituted with anti-TNP mAb incubated at the same concentrations as that for NGAL.
Antibodies Double immunofluorescence staining Affinity purified mouse monoclonal antibody (mAb) against human NGAL/Lcn2 (clone 211.1) has been described previously [32]. Rat mAb against human NGAL/ Lcn2 was purchased from R&D systems (Minneapolis, USA). mAbs against cytokeratins (CKs, clones AE1/AE3), neutrophil elastase (clone NP57), and rabbit polyclonal antibodies (pAbs) against H. pylori (code no. B0471), fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG and nonimmune rabbit IgG were purchased from Dako (Glostrup, Denmark). Cy3-conjugated goat anti-rabbit was obtained from Jackson Immunoresearch (West Grove, PA, USA). Monoclonal antibody directed against trinitrophenyl hapten (TNP, IgG1) was previously described [33].
The sections were processed as described above for the NGAL mouse mAb using heat-induced retrieval in target retrieval solution pH 6.0 (code no. S1699, Dako). The antiNGAL mouse mAb (1:150, 6.7 μg/mL) was diluted in antibody diluent (Dako) together with anti-H. Pylori pAbs (1:150) and incubated in the tissue sections for 2 h at room temperature. The antibodies were subsequently detected with FITC-conjugated goat-anti-mouse IgG, 1:200, and Cy3-conjugated goat anti-rabbit IgG, 1:200, respectively. After brief rinses with TBS, the sections were mounted with ProLong Gold antifade (Molecular Probes, Eugene, Oregon). Confocal microscopy
Immunoperoxidase staining Three micrometer thick paraffin-embedded tissue sections were deparaffinized with xylene and hydrated in gradual series of ethanol–water dilutions. For NGAL immunohistochemistry with mouse and rat mAbs, sections were heattreated in a T/T micromed microwave processor (Milestone, Sorisol, Italy) at 98°C for 30 min in target retrieval solution pH 6,0 (code no. S1699, Dako). For immunohistochemistry with antibodies against neutrophil elastase, CKs, H. pylori, and TNP, sections were pretreated with proteinase K (10 μg/mL) for 20–25 min at 37°C. Endogenous peroxidase activity was blocked by incubation in 1% hydrogen peroxide solution for 15 min and washed briefly in Tris Buffered Saline (TBS; 50 mM Tris, 150 mM NaCl, pH 7.6) containing 0.5% triton X-100. The primary antibodies were diluted in antibody diluent (Dako) and incubated for 2 h in Shandon racks (Thermo Shandon, Pittsburg, PA, USA) at the following dilutions: NGAL mouse mAb 1:200 (5.0 μg/mL), NGAL rat mAb 1:350 (1.4 μg/mL), Neutrophil Elastase 1:200, CKs 1:300, and anti-H. pylori 1:150. Subsequently, the primary antibodies were detected with EnVision reagent using either anti-mouse IgG or anti-rabbit IgG horseradish peroxidase-conjugated polymers (Dako) and polyclonal rabbit anti-rat immunoglobulins/HRP (code no. P0450, Dako). The reactions were visualized by incubating the sections with NovaRED (Vector Laboratories, Burlingame CA, USA) or DAB chromogen (Dako; for H. pylori pAbs) according to manufacturer’s instructions and counterstained with Mayer´s haematoxylin.
The double stained sections were analyzed using a confocal laser-scanning microscope, LSM 510 META (Carl Zeiss, Jena, Germany), equipped with a 488 nm argon laser and a 543 nm HeNe1 laser, as previously described [34]. Scoring for the immunoperoxidase stainings The tissue sections stained with NGAL mouse mAb from both non-neoplastic gastric mucosa adjacent to cancer tissue and from gastric cancer lesions were evaluated by two independent investigators (WAA and ODL). Neutrophils present in the microvasculature and within the tissue served as internal positive control for NGAL/Lcn2 expression. NGAL/Lcn2 immunoreactivity was scored, according to the estimated percentage of crypts showing NGAL/Lcn2positive epithelial cells, in the following four types of nonneoplastic gastric mucosa: normal mucosa, gastritis, IM, and dysplasia. The scoring was as follows: 0, less than 10% positive crypts; 1+, between 10% and 30% positive crypts; 2+, between 30% and 60% positive crypts; and 3+, more than 60% positive crypts. In gastric cancer lesions, NGAL/ Lcn2 immunoreactivity was scored based on the estimated percentage of positive cancer cells seen in the whole section. Thus, the percentages of positive cancer cells were grouped into the following categories: 0, less than 5% NGAL/Lcn2-positive cancer cells detected; 1, between 5% and 30% positively stained cells; 2, between 30% and 60% positive tumor cells; and 3, when more than 60% of cancer cells were positive.
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H. pylori positivity was scored based on the density of bacteria and the number of crypts containing bacteria into the following categories: −, no evidence of H. pylori on the section; +, less than three crypts with small clusters of bacteria; ++, either less than three crypts with dense clusters of bacteria or more than three crypts with small clusters; and +++, three or more crypts with dense clusters of bacteria. Statistical analysis χ2 analysis was performed to evaluate the differences regarding the frequency of cases having NGAL/Lcn2positive cancer cells in intestinal versus diffuse subtypes of gastric cancer. χ2 analysis and Fisher’s exact test were used to assess the correlation between NGAL/Lcn2 expression in gastric epithelial cells and H. pylori infection in normal-like and gastritis-affected mucosa. p≤0.05 was considered statistically significant in all cases.
Virchows Arch (2009) 455:225–233 Table 1 NGAL/Lcn2 immunoreactivity in non-neoplastic and neoplastic tissue sections according to the different states of the gastric mucosa and histological subtypes of gastric cancer Lesion
Normal-appearingb Gastritisb IMc Dysplasia Cancerd Intestinal Diffuse
n
6 30 17 11 38 20 18
NGAL/Lcn2 scoringa 0
1+
2+
3+
2 3 15 4 18 10 8
0 1 2 5 11 5 6
0 1 0 1 5 2 3
4 25 0 1 4 3 1
NGAL neutrophil gelatinase-associated lipocalin, Lcn2 lipocalin-2, IM intestinal metaplasia a
Scoring method is described in Material and methods section
b
Scoring according to Helicobacter pylori status is shown in Table 2
χ2 analysis for normal and gastritis vs intestinal metaplasia and dysplasia, p<0.0001 c
d
χ2 analysis for intestinal versus diffuse, p=0.84
Results NGAL/Lcn2 expression in non-neoplastic and neoplastic gastric mucosa Immunoperoxidase staining for NGAL/Lcn2 in non-neoplastic and neoplastic tissue sections demonstrated NGAL/ Lcn2 staining in all the specimens studied. We separately characterized the pattern of expression of NGAL/Lcn2 for each of the different states of the gastric mucosa: normal mucosa, gastritis, IM, dysplasia, and cancer. In general, the NGAL/Lcn2-positive cells observed in the non-neoplastic and neoplastic tissue samples included neutrophils, epithelial cells, and cancer cells. The NGAL/Lcn2 immunoreactivity of the normal mucosa (with some degree of inflammation) and gastritis-affected mucosa was intense (high frequency of sections scored as 3+), while that of IM, dysplasia, and cancer was low (high frequency of the sections scored as 0–1+; Table 1). This suggests that the expression of NGAL/Lcn2 is upregulated locally in the early steps of the gastric carcinogenesis that are characterized by chronic inflammation of the gastric mucosa. The pattern of expression of NGAL/Lcn2 varied substantially according to the level of affection of the gastric mucosa. In normal and gastritis affected mucosa, NGAL/ Lcn2 immunoreactivity was mainly seen in epithelial cells (Fig. 1a–d). NGAL/Lcn2 was, however, differentially expressed depending on the location of the epithelial cells in the crypts, with the most intense signal being observed in epithelial cells located at the neck and base of the crypts and weak or negative immunoreactivity at the surface epithelial cells (Fig. 1d). The expression of NGAL/Lcn2
was absent at foci with IM, where scattered NGAL/Lcn2positive neutrophils were seen in the lamina propria (Fig. 1c–e). In dysplastic mucosa, NGAL/Lcn2 immunoreactivity was seen in epithelial cells of the glands located deeper into the gastric mucosa (Fig. 1f). In gastric cancer lesions, NGAL/Lcn2 staining was seen in 53% (20 out of 38) of the cases in cancer cells widespread within the malignant growth, with similar frequency in intestinal (Fig. 1g) and diffuse (Fig. 1h) histological subtypes (Table 1; p=0.84; χ2 =0.04). NGAL/Lcn2 immunoreactivity in neoplasia was heterogeneous, being intense in some areas of the tumor growth and weak and/or negative in other areas (not shown). The specificity of NGAL/Lcn2 immunoreactivity was verified by analysis of a series of positive and negative controls. Similar NGAL/Lcn2 staining was obtained with mouse and rat mAbs against NGAL/Lcn2 in both nonneoplastic (Fig. 1i, j) and neoplastic tissue (not shown) in all of 15 cases tested. As negative control, we substituted the NGAL mouse mAb with a mAb against TNP. No specific staining was obtained with this antibody when incubated at similar immunoglobulin concentration as the respective anti-NGAL antibody preparation (Fig. 1k, l). H. pylori infection associates with the induction of NGAL/Lcn2 in gastric mucosa We observed, as described above, a clear and consistent NGAL/Lcn2 immunoreactivity in epithelial cells of normal mucosa and from gastritis (Table 1, Fig. 2b). Since it is known that H. pylori infection induces a chronic inflammatory
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Fig. 1 Immunoperoxidase staining for NGAL/Lcn2 in non-neoplastic and neoplastic gastric tissue. Paraffin sections from non-neoplastic (a– f, i–l) and neoplastic (g–h) gastric mucosa were incubated with mouse mAb against NGAL/Lcn2 (a–i, k), rat mAb against NGAL/Lcn2 (j), and a mAb against TNP (l). Panels i–j and k–l represent adjacent sections. In gastritis-affected mucosa (a–d), NGAL/lcn2 is expressed by epithelial cells of gastric mucosa and scattered neutrophils in the lamina propia (b; neutrophils (arrows)). NGAL/Lcn2 is seen in epithelial cells located at the neck and base of the crypts more evidently than in surface epithelial cells (d). NGAL/Lcn2 expression is absent in intestinal metaplasia (c–e), next to gastritis foci showing intense immunoreactivity (c and d; goblet cells (Gc)), where only
scattered NGAL/Lcn2-positive neutrophils are observed in the lamina propia (e; arrows). In dysplasia (f), NGAL/Lcn2 is observed in epithelial cells of glands located deep into the mucosa. In the gastric cancer lesions, NGAL/Lcn2 immunoreactivity is observed in cancer cells that are widespread within the tumor growth in both intestinal (g; stroma (St), cancer (Ca)) and diffuse subtype (h). A similar staining pattern is seen with mouse mAb (i) and rat mAb (j). As negative control, the mAb against NGAL/Lcn2 (k) was substituted with a mAb against TNP (i), and no specific staining is seen. Sections were counterstained with hematoxylin. Scale bars: ∼100 μm (a, c, i, j), ∼50 μm (d–h, k, l), ∼25 μm (b)
response in gastric mucosa [35] and that NGAL/Lcn2 expression is upregulated in response to bacterial infection and inflammation [36], we explored the possibility that the expression of NGAL/Lcn2 in epithelial cells of the gastric mucosa is associated with H. pylori infection. Tissue samples from non-neoplastic mucosa, with foci of normal and gastritis affected mucosa, were selected and stained for H. pylori.
Immunoperoxidase staining showed H. pylori in 72% (26 out of 36) of the cases. H. pylori clusters were observed at the luminal edge of the crypts, in deeper areas of the crypts, and inside some of the mucosal glands, with the highest density of bacteria in gastritis-affected mucosa (Fig. 2c). We next scored the NGAL/Lcn2 immunoreactivity in epithelial cells of the normal and gastritis-affected mucosa from adjacent
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tissue sections, as described in Material and methods, and noticed that the majority of the H. pylori-positive cases had an elevated expression of NGAL/Lcn2 (scores 2–3+) as compared to H. pylori-negative cases (Table 2). It has been shown that NGAL/Lcn2 is expressed at a basal low level in a number of human epithelial tissues such as the respiratory tract, the gastrointestinal and urinary tracts that are exposed to the external environment and thereby prone to infections [14, 18, 28], indicating that a low expression level is to be expected in mucosal surfaces. We, therefore, dichotomized the expression of NGAL/Lcn2 into NGAL/Lcn2 low immunoreactivity (scores 0, 1+) and NGAL/Lcn2 high immunoreactivity (scores 2+, 3+; Table 2) for a statistical analysis. Using this classification, we found a significant association between the expression of NGAL/Lcn2 in epithelial cells and H. pylori infection (p=0.02; χ2 =5.43; df=1; Fisher exact test p=0.04). Double immunofluorescence analysis for NGAL/ Lcn2 combined with H. pylori in non-neoplastic normal and gastritis-affected mucosa from 24 of the gastric cancer patients (18 H. pylori-positive and 6 H. pylori-negative), indeed showed intense and consistent NGAL/Lcn2 immunoreactivity in crypts where epithelial cells were in direct contact with H. pylori bacteria in the H. pylori-positive cases (Fig. 2d, f). In contrast, in H. pylori-negative cases, the NGAL/Lcn2 signal was weak in epithelial cells at similar locations (not shown).
Discussion We have investigated the expression of NGAL/Lcn2 in each of the pre-neoplastic stages leading to gastric cancer, focusing on the potential induction of this protein in response to H. pylori infection. By performing immunohistochemistry and immunofluorescence analysis, we show that NGAL/Lcn2 is upregulated in epithelial cells of normal and gastritis-affected mucosa infected with H. pylori. These are typical sites of robust inflammatory activity [7]. Interestingly, we also observed that the expression of NGAL/Lcn2 is absent at foci with IM adjacent to areas of gastritis. At IM, H. pylori infection is spontaneously eradicated and inflammation is partially resolved [2, 37]. Our results, therefore, support the role of NGAL/Lcn2 as a component of the innate immunity and suggest that H. pylori infection is causatively associated with upregulation of this molecule in gastric mucosa. In the present study, we found a significant higher NGAL/Lcn2 immunoreactivity in gastric epithelial cells of H. pylori-infected individuals than in those non-infected, which was substantiated by observing H. pylori clusters in close proximity to NGAL/Lcn2-positive epithelial cells in our double immunofluorescence analysis. NGAL/Lcn2 has been shown to be constitutively expressed in epithelial cells
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of a number of tissues that are often challenged by microorganisms such as the respiratory tract and the urinary and gastrointestinal tracts [14, 28]. NGAL/Lcn2 mRNA and protein levels are greatly increased in response to pneumococcal and mycobacterial infections in the respiratory tract and urinary tract infections, in both animal models and humans [22, 36, 38, 39]. Taking together these observations and our results, we suggest that the constitutive basal expression of NGAL/Lcn2 that is observed in the gastric epithelium ([14, 28] and this paper) is substantially upregulated upon H. pylori infection. A recent study in rhesus macaques, showing that NGAL/Lcn2 is induced in gastric mucosa of monkeys challenged with certain H. pylori strains [30], is in agreement with our observations and suggests that the upregulation of NGAL/Lcn2 by H. pylori may be related to specific bacterial virulence factors. A particularly interesting finding of this study was the abrupt down-regulation of NGAL/Lcn2 expression observed at IM foci located next to normal and gastritis H. pylori-infected mucosa showing intense NGAL/Lcn2 immunoreactivity. A number of histological and physiological changes occur in the gastric epithelium during the pre-neoplastic stages leading to gastric cancer, including a focal loss of glands and specialized cells (atrophic gastritis), followed by the transformation of the stomach mucosa into an intestinal-like epithelium (IM) [8, 40]. These changes are not well tolerated by H. pylori and results in its spontaneous disappearance from the transformed epithelium [37, 41]. Accordingly, we did not observe H. pylori bacteria at intestinal metaplastic glands. It is, therefore, tempting to suggest that the abolition of NGAL/Lcn2 expression on IM is a consequence of the natural eradication of H. pylori occurring at these foci. Thus, our findings in IM argue in favor of the potential link between H. pylori infection and the upregulation of NGAL/Lcn2 in the gastric epithelium. Iron is an essential element for the growth and virulence of H. pylori, and the general consensus is that H. pylori obtains this element from human molecules, mainly lactoferrin and heme [27, 42–44]. Despite one study suggesting the production of siderophores by H. pylori [45], most investigations have been unable to reproduce that finding or to demonstrate the use of exogenous siderophores as a source of iron by this bacterium [44, 46]. Nevertheless, the induction of NGAL/Lcn2 is not restricted to microorganisms that produce or uptake siderophores. The induction of NGAL/Lcn2 has been consistently linked to inflammation. High NGAL/Lcn2 mRNA and protein levels have been found in epithelial cells in inflammatory colorectal diseases such as inflammatory bowel disease, appendicitis [18], as well as in other processes associated with intense inflammatory activity [20, 47]. As part of the inflammatory response against H. pylori infection, a complex set of inflammatory mediators are released by
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Fig. 2 Immunohistochemical analyses of NGAL/Lcn2 and H. pylori in non-neoplastic gastric mucosa. Sections from samples with gastric mucosa adjacent to the neoplastic lesion were processed for immunoperoxidase staining for NGAL/Lcn2 (a, b), H. pylori (c), or for immunofluorescence analysis (d–f) using mAb against NGAL/ Lcn2, pAbs against H. pylori, and NGAL mAb together with H. pylori pAbs, respectively. NGAL/Lcn2 immunoreactivity is weak, if observed, in H. pylori-negative mucosa (a). In H. pylori-infected mucosa, NGAL/ Lcn2 is strongly expressed on epithelial cells (b). H. pylori clusters (red stars) can be observed at the luminal side in some of the crypts along
the gastric epithelium with the highest density in gastritis-affected mucosa (c). In the double immunofluorescence analysis, mAb against NGAL/Lcn2 together with pAbs against H. pylori (double staining in d–f) were detected with FITC-conjugated goat antimouse (green fluorescence) and Cy3-conjugated goat antirabbit (red fluorescence), respectively. Intense NGAL/Lcn2 immunoreactivity is seen in epithelial cells that are in direct contact with H. pylori bacteria (d-f). Nomarsky DIC imaging technique was used in d–f. Scale bars: ∼100 μm (a, b), ∼12 μm (c), ∼20 μm (d–f)
epithelial and infiltrating inflammatory cells into the gastric mucosa, including IL-1β and NF-κβ [48]. Certain H. pylori virulence factors have been shown to potentiate this inflammatory response [49]. It has been demonstrated that IL-1β and NF-κβ (stabilized by its cofactor IκB-ζ) play a central role in the induction of NGAL/Lcn2 in epithelial cells [19, 50]. Thus, it is likely that the upregulation of NGAL/
Lcn2 by H. pylori in gastric epithelial cells is driven by mediators of inflammation being released into the gastric epithelium in response to the bacterial infection. It was recently demonstrated in animal models (rhesus macaques and mice) that pathogenic strains of Salmonella enterica induce NGAL/Lcn2 in colonic mucosa, which confers a competitive advantage to the bacterium for colonizing and growing in the inflamed intestine [51]. In agreement to this view, Hornsby et al. [30] hypothesized that the induction of a set of antimicrobial molecules, including NGAL/Lcn2, in rhesus monkeys infected with H. pylori may indeed serve to increase the competitive advantage of this bacterium. We, therefore, do not exclude the possibility that high expression of NGAL/Lcn2 in gastric mucosa may facilitate H. pylori infection. Other functions have been suggested for NGAL/Lcn2 in addition to its antimicrobial role [52]. NGAL/Lcn2 has been shown to be expressed on neoplastic cells in several types of adenocarcinomas including breast, pancreas, colon, and ovarian cancer [28, 53–56], and elevated protein levels have been correlated with poor prognosis in some of these malignancies [56, 57]. In the current study, we observed
Table 2 NGAL/Lcn2 immunoreactivity in epithelial cells in relation to the Helicobacter pylori status in non-neoplastic tissue sections diagnosed as normal-appearing and gastritis-affected mucosa n
H. pylori negativea H. pylori positivea Total
10 26 36
NGAL/Lcn2 scoring 0
1+
2+
3+
3 2 5
1 0 1
1 0 1
5 24 29
NGAL neutrophil gelatinase-associated lipocalin, Lcn2 lipocalin-2 a
Fisher’s exact test for H. pylori-positive versus H. pylori-negative, p=0.04
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NGAL/Lcn2 immunoreactivity in gastric cancer cells in one half of the studied cases, with similar frequency in both intestinal and diffuse gastric cancer histological subtypes. A recent study found that NGAL/Lcn2 levels in tissue and urine from breast cancer patients are associated with invasion and metastasis and demonstrated, in cell lines transfected with NGAL/Lcn2, that this molecule promotes breast cancer progression by inducing epithelial to mesenchymal transition [58]. The molecular mechanisms explaining the role of NGAL/Lcn2 in carcinogenesis remain entirely unknown, but it has been speculated that expression of NGAL/Lcn2 provides the carcinoma cells the ability to acquire iron and thus, supporting their growth [52, 59]. Therefore, the expression of NGAL/Lcn2 in gastric cancer in connection to progression and prognosis of the disease should be further investigated. Acknowledgements We thank Ms. Öznur Turan and Ms. Agnieszka Ingvorsen for their excellent technical assistance and Mr. John Post for his photographic assistance. This study was supported by the Danish Cancer Society, the Lundbeck Foundation, Haukeland University Hospital (Helse-Vest), and Vicerrectoría de Investigación of the University of Costa Rica. Conflict of interest statement of interest.
We declare that we have no conflict
References 1. Parkin DM, Bray FI, Devesa SS (2001) Cancer burden in the year 2000. The global picture. Eur J Cancer 37(Suppl 8):S4–S66 2. Stemmermann GN, Fenoglio-Preiser C (2002) Gastric carcinoma distal to the cardia: a review of the epidemiological pathology of the precusors to a preventable cancer. Pathology 34:494–503 3. Correa P (1992) Human gastric carcinogenesis: a multistep and multifactorial process—First American Cancer Society Award Lecture on cancer epidemiology and prevention. Cancer Res 52:6735–6740 4. Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S et al (2001) Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 345:784–789 5. Huang JQ, Zheng GF, Sumanac K, Irvine EJ, Hunt RH (2003) Meta-analysis of the relationship between cagA seropositivity and gastric cancer. Gastroenterology 125:1636–1644 6. Wilson KT, Crabtree JE (2007) Immunology of Helicobacter pylori: insights into the failure of the immune response and perspectives on vaccine studies. Gastroenterology 133:288–308 7. Robinson K, Argent RH, Atherton JC (2007) The inflammatory and immune response to Helicobacter pylori infection. Best Pract Res Clin Gastroenterol 21:237–259 8. Correa P, Houghton J (2007) Carcinogenesis of Helicobacter pylori. Gastroenterology 133:659–672 9. Fox JG, Wang TC (2007) Inflammation, atrophy, and gastric cancer. J Clin Invest 117:60–69 10. Neilands JB (1995) Siderophores: structure and function of microbial iron transport compounds. J Biol Chem 270:26723–26726 11. Braun V, Braun M (2002) Active transport of iron and siderophore antibiotics. Curr Opin Microbiol 5:194–201 12. Krewulak KD, Vogel HJ (2008) Structural biology of bacterial iron uptake. Biochim Biophys Acta 1778:1781–1804
13. Kjeldsen L, Johnsen AH, Sengelov H, Borregaard N (1993) Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem 268:10425–10432 14. Cowland JB, Borregaard N (1997) Molecular characterization and pattern of tissue expression of the gene for neutrophil gelatinase-associated lipocalin from humans. Genomics 45:17– 23 15. Kjeldsen L, Cowland JB, Borregaard N (2000) Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse. Biochim Biophys Acta 1482:272–283 16. Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN et al (2002) The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 10:1033–1043 17. Holmes MA, Paulsene W, Jide X, Ratledge C, Strong RK (2005) Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration. Structure 13:29–41 18. Nielsen BS, Borregaard N, Bundgaard JR, Timshel S, Sehested M et al (1996) Induction of NGAL synthesis in epithelial cells of human colorectal neoplasia and inflammatory bowel diseases. Gut 38:414–420 19. Cowland JB, Sorensen OE, Sehested M, Borregaard N (2003) Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha. J Immunol 171:6630–6639 20. Sorensen OE, Cowland JB, Theilgaard-Monch K, Liu L, Ganz T et al (2003) Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes, a consequence of common growth factors. J Immunol 170:5583–5589 21. Berger T, Togawa A, Duncan GS, Elia AJ, You-Ten A et al (2006) Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury. Proc Natl Acad Sci U S A 103:1834–1839 22. Chan YR, Liu JS, Pociask DA, Zheng M, Mietzner TA et al (2009) Lipocalin 2 is required for pulmonary host defense against Klebsiella infection. J Immunol 182:4947–4956 23. Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA et al (2004) Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432:917–921 24. van Amsterdam K, van Vliet AH, Kusters JG, van der Ende A (2006) Of microbe and man: determinants of Helicobacter pylorirelated diseases. FEMS Microbiol Rev 30:131–156 25. Velayudhan J, Hughes NJ, McColm AA, Bagshaw J, Clayton CL et al (2000) Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter. Mol Microbiol 37:274–286 26. Waidner B, Greiner S, Odenbreit S, Kavermann H, Velayudhan J et al (2002) Essential role of ferritin Pfr in Helicobacter pylori iron metabolism and gastric colonization. Infect Immun 70: 3923–3929 27. Perez-Perez GI, Israel DA (2000) Role of iron in Helicobacter pylori: its influence in outer membrane protein expression and in pathogenicity. Eur J Gastroenterol Hepatol 12:1263–1265 28. Friedl A, Stoesz SP, Buckley P, Gould MN (1999) Neutrophil gelatinase-associated lipocalin in normal and neoplastic human tissues. Cell type-specific pattern of expression. Histochem J 31:433–441 29. Playford RJ, Belo A, Poulsom R, Fitzgerald AJ, Harris K et al (2006) Effects of mouse and human lipocalin homologues 24p3/ lcn2 and neutrophil gelatinase-associated lipocalin on gastrointestinal mucosal integrity and repair. Gastroenterology 131:809– 817 30. Hornsby MJ, Huff JL, Kays RJ, Canfield DR, Bevins CL et al (2008) Helicobacter pylori induces an antimicrobial response in
Virchows Arch (2009) 455:225–233
31. 32.
33.
34.
35. 36. 37.
38.
39.
40.
41.
42.
43.
44.
45.
rhesus macaques in a cag pathogenicity island-dependent manner. Gastroenterology 134:1049–1057 Japanese Gastric Cancer A (1998) Japanese classification of gastric carcinoma - 2nd english edition. Gastric Cancer 1:10–24 Kjeldsen L, Koch C, Arnljots K, Borregaard N (1996) Characterization of two ELISAs for NGAL, a newly described lipocalin in human neutrophils. J Immunol Methods 198:155–164 Boulianne GL, Hozumi N, Shulman MJ (1984) Production of functional chimaeric mouse/human antibody. Nature 312:643– 646 Nielsen BS, Rank F, Illemann M, Lund LR, Dano K (2007) Stromal cells associated with early invasive foci in human mammary ductal carcinoma in situ coexpress urokinase and urokinase receptor. Int J Cancer 120:2086–2095 Atherton JC (2006) The pathogenesis of Helicobacter pyloriinduced gastro-duodenal diseases. Annu Rev Pathol 1:63–96 Xu S, Venge P (2000) Lipocalins as biochemical markers of disease. Biochim Biophys Acta 1482:298–307 Valle J, Kekki M, Sipponen P, Ihamaki T, Siurala M (1996) Longterm course and consequences of Helicobacter pylori gastritis. Results of a 32-year follow-up study. Scand J Gastroenterol 31: 546–550 Nelson AL, Barasch JM, Bunte RM, Weiser JN (2005) Bacterial colonization of nasal mucosa induces expression of siderocalin, an iron-sequestering component of innate immunity. Cell Microbiol 7:1404–1417 Saiga H, Nishimura J, Kuwata H, Okuyama M, Matsumoto S et al (2008) Lipocalin 2-dependent inhibition of mycobacterial growth in alveolar epithelium. J Immunol 181:8521–8527 Gutierrez-Gonzalez L, Wright NA (2008) Biology of intestinal metaplasia in 2008: more than a simple phenotypic alteration. Dig Liver Dis 40:510–522 Kokkola A, Kosunen TU, Puolakkainen P, Sipponen P, Harkonen M et al (2003) Spontaneous disappearance of Helicobacter pylori antibodies in patients with advanced atrophic corpus gastritis. APMIS 111:619–624 Dhaenens L, Szczebara F, Husson MO (1997) Identification, characterization, and immunogenicity of the lactoferrin-binding protein from Helicobacter pylori. Infect Immun 65:514–518 Worst DJ, Otto BR, de Graaff J (1995) Iron-repressible outer membrane proteins of Helicobacter pylori involved in heme uptake. Infect Immun 63:4161–4165 Husson MO, Legrand D, Spik G, Leclerc H (1993) Iron acquisition by Helicobacter pylori: importance of human lactoferrin. Infect Immun 61:2694–2697 Illingworth DS, Walter KS, Griffiths PL, Barclay R (1993) Siderophore production and iron-regulated envelope proteins of Helicobacter pylori. Zentralbl Bakteriol 280:113–119
233 46. Dhaenens L, Szczebara F, Van Nieuwenhuyse S, Husson MO (1999) Comparison of iron uptake in different Helicobacter species. Res Microbiol 150:475–481 47. Sorensen OE, Thapa DR, Roupe KM, Valore EV, Sjobring U et al (2006) Injury-induced innate immune response in human skin mediated by transactivation of the epidermal growth factor receptor. J Clin Invest 116:1878–1885 48. Algood HM, Cover TL (2006) Helicobacter pylori persistence: an overview of interactions between H. pylori and host immune defenses. Clin Microbiol Rev 19:597–613 49. Brandt S, Kwok T, Hartig R, Konig W, Backert S (2005) NFkappaB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein. Proc Natl Acad Sci U S A 102:9300–9305 50. Cowland JB, Muta T, Borregaard N (2006) IL-1beta-specific upregulation of neutrophil gelatinase-associated lipocalin is controlled by IkappaB-zeta. J Immunol 176:5559–5566 51. Raffatellu M, George MD, Akiyama Y, Hornsby MJ, Nuccio SP et al (2009) Lipocalin-2 resistance confers an advantage to Salmonella enterica serotype Typhimurium for growth and survival in the inflamed intestine. Cell Host Microbe 5:476–486 52. Clifton MC, Corrent C, Strong RK (2009) Siderocalins: siderophorebinding proteins of the innate immune system. Biometals 22:557– 564 53. Stoesz SP, Friedl A, Haag JD, Lindstrom MJ, Clark GM et al (1998) Heterogeneous expression of the lipocalin NGAL in primary breast cancers. Int J Cancer 79:565–572 54. Furutani M, Arii S, Mizumoto M, Kato M, Imamura M (1998) Identification of a neutrophil gelatinase-associated lipocalin mRNA in human pancreatic cancers using a modified signal sequence trap method. Cancer Lett 122:209–214 55. Lim R, Ahmed N, Borregaard N, Riley C, Wafai R et al (2007) Neutrophil gelatinase-associated lipocalin (NGAL) an earlyscreening biomarker for ovarian cancer: NGAL is associated with epidermal growth factor-induced epithelio-mesenchymal transition. Int J Cancer 120:2426–2434 56. Bauer M, Eickhoff JC, Gould MN, Mundhenke C, Maass N et al (2008) Neutrophil gelatinase-associated lipocalin (NGAL) is a predictor of poor prognosis in human primary breast cancer. Breast Cancer Res Treat 108:389–397 57. Devarajan P (2007) Neutrophil gelatinase-associated lipocalin: new paths for an old shuttle. Cancer Ther 5:463–470 58. Yang J, Bielenberg DR, Rodig SJ, Doiron R, Clifton MC et al (2009) Lipocalin 2 promotes breast cancer progression. Proc Natl Acad Sci U S A 106:3913–3918 59. Devireddy LR, Gazin C, Zhu X, Green MR (2005) A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake. Cell 123:1293–1305