Gut 1996; 38: 414-420

Induction ofNGAL synthesis in epithelial cells ofhuman colorectal neoplasia and inflammatorybowel diseases

B S Nielsen, N Borregaard, J R Bundgaard, S Timshel, M Sehested, L Kjeldsen

AbstractIn inflammatory and neoplastic disordersof the colon a defect barrier function ofthe mucosa may result in absorption ofbacterial products from the intestinallumen. These products may furtherrecruit inflammatory cells and thus aug-ment the inflammatory response. A novellipocalin in neutrophils, neutrophil gelati-nase associated lipocalin (NGAL), withthe ability to bind bacterial formyl-peptides, has been described and there-fore it is of interest to investigate theexpression of this protein in diseases ofthe colon. Expression ofNGAL was inves-tigated by immunohistochemistry and bymRNA in situ hybridisation in normalcolon and in neoplastic and inflammatorycolorectal diseases. A very high expres-sion ofNGAL was seen in colonic epithe-lium in areas of inflammation, both innon-malignant epithelium (diverticulitis,inflammatory bowel disease, and appen-dicitis) as well as in premalignant andmalignant neoplastic lesions of the colon.In adenocarcinoma, the NGAL expressionwas especially abundant in the transi-tional mucosa and in the superficial ulcer-ated area. On the other hand, no NGALexpression could be detected in lymphnode metastases from these adenocarci-nomas. A weak expression of NGAL insome epithelial cells was only occasionallyseen in normal colon. In conclusion,NGAL synthesis is induced in epithelialcells in inflammatory and neoplastic,colorectal diseases. NGAL may serve animportant anti-inflammatory function asa scavenger ofbacterial products.(Gut 1996; 38: 414-420)

Keywords: in situ hybridisation,immunohistochemistry, neutrophils, lipocalin,inflammation.

Inflammatory bowel diseases are characterisedby a prominent infiltration of the mucosa byinflammatory cells including macrophages,lymphocytes, plasma cells, and neutrophils.The cellular infiltration is especially abundantaround ulcerations or fissures of the bowel wall.Ulceration is also a characteristic feature ofadenocarcinoma of the colon, in which acuteinflammation with infiltration of macrophagesand neutrophils is seen. The role of cytokines ininflammatory bowel disease has been thesubject of intense investigation over recent

years. In inflammatory bowel disease, synthesisof interleukin 1 (IL1), tumour necrosisfactor cx (TNFot), and IL2 in macrophages andlymphocytes may be induced as a result ofpassage through the colonic mucosa ofbacterial products, including lipopolysaccha-ride.1-6 These cytokines may in turn inducesynthesis of a variety of other inflammatorymediators including prostaglandins, plateletactivating factor, leukotrienes, IL8, granulocytecolony stimulating factor (G-CSF),l 5 7-11 mostofwhich are chemotactic for neutrophils, whichare then recruited to and activated in theinflammatory focus. This may induce tissuedestruction and ulceration, and further impairthe barrier function of the mucosa.12 The roleof the mucosa itself in the inflammatory reac-tion of inflammatory bowel disease and coloncancer has become increasingly evident.13 Inresponse to local inflammation/inflammatorymediators, colonic epithelial cells (includingcancer cell lines) are induced to expressimmunologically active molecules like MHCclass II,14-16 the adhesion protein intercellularadhesion molecule- 1,14 17 the cytokines IL8,GM-CSF, MCP-1, and TNFo, 10819prostanoids,7 20 and 12-lipoxygenase, whichparticipates in the formation of leukotrienesfrom arachidonic acid.2'We have described the isolation and charac-

terisation of a novel protein in human neutro-phils, neutrophil gelatinase associated lipocalin(NGAL).22 By northern blotting of a variety oftissues including brain, lung, heart, kidney,liver, blood, and bone marrow cells, we onlyfound mRNA expression in neutrophil pre-cursors in the bone marrow.23 Furthermore,NGAL was shown to exist both as a 25 kDamonomer, as a 46 kDa homodimer, andin a covalent complex with neutrophil gelati-nase.22 24 As the name implies, NGAL is amember of the lipocalin family. These glyco-proteins all have a protein backbone of 18 to20 kDa.25 26 Despite a low degree of overallamino acid identity, the lipocalins share certainconserved motifs, which are responsible fortheir common tertiary structure consisting ofone ox helix and an eight stranded 1 barrelsurrounding a hydrophobic core.26-28 Thehydrophobic core has been described as apocket that enables the lipocalins to bind smalllipophilic substances. Rather than beingspecific for one ligand, it has been postulatedthat many lipocalins might bind severaldifferent lipophilic substances.25 We haveshown that NGAL can bind a radioiodinatedderivative of the bacterial chemotactic formyl-peptide FMLP,26 and it is possible that

Department ofPathology, SundbyHospital, DenmarkB S NielsenS TimshelM Sehested

Finsen LaboratoryB S Nielsen

Granulocyte ResearchLaboratory,Department ofHematologyN BorregaardL Kjeldsen

Department of ClinicalBiochemistryJ R Bundgaard

University Hospital,Rigshospitalet,Copenhagen, Denmark

Correspondence to:Professor Niels Borregaard,Granulocyte ResearchLaboratory, Department ofHematology, L-4042,University Hospital,Rigshospitalet, Blegdamsvej9, 2100 Copenhagen 0,Denmark.

Accepted for publication7 September 1995

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Expression ofNGAL in colorectal diseases

Expression ofNGAL mRNA detected by in situhybridisation in colorectal lesions

Colorectal sion Total examined Positive

Normal epithelium* 13 4§Appendix (normal)t 9 4§Appendix (inflamed) 8 8Diverticulitis 5 4Ulcerative colitis 3 3Crohn's disease 5 5Adenoma 2 2Adenocarcinoma 11 10Lymph node metastasis* 3 0

*Obtained from patients with adenocarcinoma (sevenspecimens) and from patients suffering from ulcerative colitisand Crohn's disease (six specimens), tobtained athysterectomy, *from primary colon cancers that expressedNGAL. §0nly weak staining was seen.

NGAL also binds other lipophilic inflammatorymediators like platelet activating factor,leukotriene B4, and lipopolysaccharide. NGALtherefore may possess important immuno-modulatory actions.One way of obtaining knowledge about the

function of a protein is to study its localisationin various organs and diseases. A number ofproinflammatory mediators are induced ininflammatory bowel diseases and presumablyalso in adenocarcinoma of the colon. Further-more, a defect barrier function of the mucosa

in these disorders potentially leads to thetransepithelial passage of lipopolysaccharideand bacterial formyl-peptides. As NGAL mayhave the ability to bind these substances, we

found it ofinterest to investigate the expressionof NGAL in inflammatory bowel disease andneoplastic lesions of the colon. This was doneby immunohistochemistry and mRNA in situhybridisation.

Methods

TissuesSpecimens from 14 colorectal adenocarcino-mas containing both malignant and normalepithelium were used for immunohisto-chemistry and 11 of these also for in situhybridisation. The Table lists the additionalspecimens of different inflammatory boweldiseases used for in situ hybridisation. Allspecimens were obtained at surgery.For optimal fixation of tissue for immuno-

histochemistry, specimens were fixed for onlyone hour in 4% phosphate buffered formalinfollowed by ethanol dehydration/fixation andparaffin wax embedding. Prolonged fixation ofspecimens caused loss of NGAL immuno-reactivity. These short-term fixed specimenscompletely lacked detectable mRNA, however,as determined by in situ hybridisation with a

28'mer thymidine probe for the poly-A tail ofmRNAs (see later). On the other hand, therewas a strong mRNA signal in 24 hour formalinfixed specimens using the poly-T probe.Similar findings with absence of in situhybridisation signal in ethanol fixed specimensbut increased mRNA signal and reducedinmmunoreactivity after prolonged formalinfixation have been described by Urieli-Shoval etal.30 For optimal fixation of tissues for in situhybridisation specimens were therefore fixed

for 24 hours in 4% phosphate buffered formalinand subsequently embedded in paraffin wax.

ImmunohistochemistryFive ,um sections were cut and air driedovernight. Paraffin wax was dissolved in 60°Ccocoa oil for 20 minutes and sections were thenhydrated through decreasing concentrations ofethanol. Sections were trypsinised (0.05%trypsin (Merck, Darmstadt, Germany)) in50 mM TRIS, 0.1% CaCl2 (w/v), pH 74,followed by blocking using 10% normal swineserum (X-901, Dako, Glostrup, Denmark).Incubation of affinity purified polyclonal rabbitanti-NGAL antibodies (0 5 mg/m122) wasperformed at 500 to 2000-fold dilution in TRISbuffered saline (TBS), pH 7-6, containing0-25% bovine serum albumin (BSA). Sectionswere incubated at 40C overnight followed byincubation at room temperature for anadditional 30 to 60 minutes. Primary antibodieswere detected by biotinylated swine antirabbitimmunoglobulin antibodies followed by incuba-tion with the ABComplex/HRP (according toinstructions given in the ABC kit from Dako).All steps were followed by three washes in TBS.Sections were developed with 3-amino-9-ethyl-carbazol (Sigma Chemical, St Louis, MO) for10 minutes and counterstained with haema-toxylin. Pre-immune rabbit IgG (Dako, X-903)was used as a negative control. In addition,absorbed anti-NGAL (adding 10-fold molarexcess of purified NGAL to the anti-NGALantibodies) was used as a control of the speci-ficity of the reaction.

In situ hybridisationNGAL cDNA was subcloned in pBluescript(Stratagene) and sequenced as described.23Antisense and sense 35S labelled RNA probeswere obtained by in vitro transcription andsubsequently used for in situ hybridisationusing the method described by Pyke et al.31Briefly, 35S UTP labelled antisense and senseriboprobes were diluted to a final activity of106 cpm/,u. Four gl were used for hybridisa-tion for each section. Hybridisation wasperformed overnight at 50°C. Sections weresubsequently exposed on an autoradiographicemulsion for 10-14 days before development.For staining of total mRNA pretreatment oftissue sections was performed as above.The 92-3T-Dig Poly-T probe was applied(5 ng/ml) in 50% formamid, 12-5% dextransulphate, 1 mg/ml tRNA, Ficoll 400 (0-02%(wt/vol)), polyvinylpyrolidone (0.02% (wlv)),TRIS-HCI (10 mM, pH 6.8), and EDTA(0 5 mM). Sections were incubated for onehour followed by washes with SSC-buffersas follows: 2XSSC (0 3 M sodium chloride,0-03 M sodium citrate, pH 7.0), 1 XSSC, and0-5xSSC each for 30 minutes. All steps wereperformed at room temperature. Afterwashing in TBS-Triton, sections wereblocked for unspecific binding with 0-25%normal rabbit serum for 10 minutes. Sectionswere then incubated with alkaline phos-phatase conjugated mouse antidigoxigenin

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(Boehringer Mannheim no 1093 274,Mannheim, Germany) diluted 1:500 in TBS-BSA and finally developed for 30 minuteswith nitroblue tetrazoleum/5-bromo-4-chloro-3-indolylphosphate (Sigma Chemical, St Louis,MO).

Results

Expression ofNGAL in colorectal neoplasiaImmunohistochemistry - in all 14 cases of

adenocarcinoma an intense staining was seenin both normal and malignant epithelial cellsin the transitional mucosa, and in the malig-nant epithelial cells just below the ulcer (Fig1 a). No or very few epithelial cells werepositive in the adjacent normal appearingepithelium of the same specimens and in theinvasive area of the tumour. Intense NGALimmunostaining was seen in all neutrophils asexpected. These were found located in vesselsand as tissue infiltrating neutrophils in thetumour and in the ulcer (Fig 1 a).

In situ hybridisation was performed on 11 of14 tumours investigated by immunohistochem-istry. These samples also contained bothnormal and malignant epithelium, but differedfrom the samples used for immunohisto-chemistry in the fixation procedure (seeMethods). A strong in situ hybridisation signalwas seen in 10 of 1 1 cases (Table and Fig lband le). In these 10 cases, hybridisation wasconfined to epithelial cells localised to a patternidentical to the pattern seen by immunohisto-chemistry - that is, in cancer cells below theulcer (Fig lb) and in normal and malignantcells in the transitional mucosa (Fig 1 e). In con-trast with the immunohistochemical localisa-tion, no in situ hybridisation signal was seen inany neutrophils. Cancer cells located in thedeeper part of the tumour and at the invasivefront were usually negative. At a distance of upto 1 cm from the tumour a few crypts of thenormal epithelium were weakly positive in someofthe cases. Furthermore, we investigated threelymph node metastases, which were obtainedfrom specimens from patients with NGALexpressing primary tumours. These were allnegative. In colonic adenomas, NGAL mRNAexpression was seen only in the epithelial cellson the luminal surface of the lesions (Fig if).

Expression ofNGAL mRNA in variousinflammatory diseases of colon and in appendicitisThe epithelial cells of virtually all specimens ofdiverticulitis (Fig 2a and 2b), ulcerative colitis(Fig 2c and 2d), and inflamed specimens ofCrohn's disease (even with low grade inflam-mation as seen in Fig 2e and 2f) showed strongNGAL expression (Table). On the other hand,all epithelial cells of unaffected colorectal spec-imens from these patients with inflammatorybowel disease or diverticulitis were negative forNGAL expression (not shown). No signal wasseen in infiltrating neutrophils.A strong signal was seen in the epithelial

cells in all the inflamed appendixes investi-gated (Table, Fig 2g).

Expression ofNGAL mRNA in normal colon andappendicitisNGAL mRNA was detected at a low level incrypts of four of 14 cases of histologicallynormal colonic epithelium from patients withinflammatory bowel disease or colorectalcancer, and in four of nine cases of normalappendix (Table). No differences regardingcellular infiltration were seen in the positivespecimens compared with the negative ones.Scattered neutrophils present in normal colonwere strongly positive by immunohisto-chemistry, but negative by in situ hybridisa-tion. In addition, mast cells were positive byimmunohistochemistry, but stained muchweaker than neutrophils.

Control experimentsAs negative controls in immunohistochemistrywe used pre-immune rabbit IgG or absorbedantibodies (see Methods). No signal was seenshowing that no unspecific binding of primaryantibodies occurred and that endogenous per-oxidase activity was blocked (Fig 1 c). As a neg-ative control for the in situ hybridisation, weapplied the antisense and the sense probe onadjacent sections. The sense probe was alwaysnegative (Fig 1d).

DiscussionInduction of NGAL synthesis is a striking andconsistent finding in epithelial cells in neo-plastic and inflammatory disorders of colonand appendix, as shown by a very strongsignal for NGAL by in situ hybridisation inmost specimens of affected tissue (32 of 34specimens), and furthermore by immuno-histochemical staining of epithelial cells ofcolorectal cancers (all 14 cases investigated).The combined use of in situ hybridisation andimmunohistochemistry and the identical stain-ing pattern seen in epithelial cells provideevidence that the increased mRNA expres-sion of NGAL in epithelial cells is actuallyaccompanied by production of mature protein.Although we found an expression ofmRNA forNGAL in epithelial cells in four of nine speci-mens of normal appendixes and in four of 13sections of normal colon by in situ hybridisa-tion, this expression was weak and matureprotein could not be detected by immunohisto-chemistry.As expected, neutrophils present in both

normal and inflamed colon contain NGAL asshown by a strong immunohistochemical stain-ing of these cells. No mRNA for NGAL waspresent in neutrophils, showing that matureneutrophils in the colonic mucosa do notactively synthesise NGAL despite the potentialpresence of inflammatory mediators like inter-feron v and TNFa., which have been shown toinduce the synthesis of a variety of proteins inmature neutrophils.32-34 This implies thatNGAL, present in neutrophils in colonicmucosa, is synthesised in immature neutrophilprecursors in the bone marrow,23 where NGALis packed in specific granules.35 It is clear thatthe induction of NGAL synthesis in colon

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Expression ofNGAL in colorectal diseases

Figure 1: NGAL proteinand mRNA detected byimmunohistochemistry andin situ hybridisation inneoplastic colorectal lesions.Immunohistochemicalstaining ofanadenocarcinoma (a) showsNGAL expression insuperficial cancer cellsbelow the ulcer (largearrows in (a), ulcer markedby (u)). In addition,neutrophils present in theulcer and in the stroma(small arrows) are stronglystained. The reactivity ofthe staining is specific asshown in (c), which showslack ofstaining, when pre-immune rabbit antibodieswere applied in an adjacentsection to (a). Cancer celsbelow the ulcer were alsostrongly stained by mRNAin situ hybridisation(arrows in (b)). Inaddition, cancer cells in thetransitional mucosa werepositive (arrow in (e)). Noin situ hybridisation signalis seen in adjacent normal(n) appearing epithelium in(e) and (I). Likewise,neutrophils present in theulcer and in the stromawere negative. Appropriatecontrols for in situhybridisation were negativeas shown in (d), whichshows lack ofhybridisationwith the sense probe in asection adjacent to (b). Inadenoma (f), NGALmRNA expression was onlyseen in epithelial cells on theluminal surface of the lesion(arrows). Magnifications:(a)-(d): X40, (e): X10,and W0: X4.

a -

f

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Figure 2: In situhybridisation forNGALmRN4 in variousinflammatory diseases ofcolon and appendix.Photomicrographs showdivericulitis ((a): brightfield and (b): dark field),ulertve colitis ((c):brightfield and (d): darkfield), Crohn's disease((e): brightfield and(9: dark field,magnfication oflower leftcorner of (e), andappendicitis (g)). A strongsignaforNGAL mRNA isseen in the epiheium ofaffected tissue. Note thecharacteristc cryptabscesses in the centre of thephoomicrographs (c) and(d). Magnification:(a)-(d) and (9 X200,(e) and (g) X40.

epithelium is local, as it is seen in the affectedtissue only. Furthermore, the induction is notspecific for any particular colorectal disease butseems to be a response to inflammation ingeneral, as inflammation is a common featureof inflammatory bowel disease, diverticulitis,appendicitis, and adenocarcinoma of the colon.The importance of the inflammatory response

for induction of NGAL synthesis is under-scored by the fact, that no NGAL synthesiscould be detected in lymph-node metastasesfrom NGAL expressing primary tumours. Thisshows that the ability to synthesise NGAL isnot part of the neoplastic process, but isinduced as a result of the presence ofinflammatory mediators liberated from the

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Lumen0e

Epithelium

Lamina

itrophil prmia

* ~ Formyl-peptides@ @ LPS

, V NGAL

'Enhancement' of inflammatory response

NGAL expressionin epithelial cells

v V 'v

.. , . ... v

'Downregulation' of inflammatory response

Figure 3: Schematic presentation of the putative function ofNGAL expressed in epithelial cells during inflammation. Tothe left is shown two epithelial cells, where a defect barrierfunction permits the transepithelial passage oflipopolysaccharideandformyl-peptides from the lumen to the lamina propria. Lipopolysaccharide induces the synthesis in macrophages ofTNFa and IL1, which in turn may lead to production of mediators like G-CSF, LTB4, PAF, and IL 8. '5 7-11 Thesemediators will, in concert with formyl-peptides, attract and activate neutrophils, which may result in augmented tissuedamage. To the right are two epithelial cells that synthesise NGAL. NGAL is presumably liberated to the intestinal lumenwhere it binds lipopolysaccharide andformyl-peptides, thus preventing these substances from recruiting and activatingmacrophages and neutrophils. The NGAL production in epithelial cells may therefore downregulate the inflammatoryresponse. Other cells participating in the inflammatory reaction are omittedfor reasons ofsimplicity. Likewise, noneutrophils are shown on the luminalface of the epithelium, although NGAL liberatedfrom these may act in conjunctionwith NGALfrom epithelial cells in the binding of bacterial products.

local environment. The mechanisms underly-ing induction ofNGAL synthesis are unknown,but the presence of NGAL in only the mostsuperficial parts of adenomas and adenocarci-nomas suggests that the initiating agentsoriginate in the intestinal lumen. One candidateis lipopolysaccharide, which has been shown toinduce the synthesis of the NGAL analogue24p3 in cultured murine macrophages.36Many cytokines and inflammatory mediators

that are induced in various inflammatory statesofthe colon (including IL1, TNFoc, IL2, plateletactivating factor, and G-CSF1A 8) are producedby 'traditional' inflammatory cells like lympho-cytes, macrophages, and neutrophils, but severalreports have shown that the epithelial cellsalso take part in the inflammatory reaction.13 19An array of substances like MHC classIIJ14-16 ICAM-1,14 17 various proinflammatorycytokines,10 18 19 prostanoids,7 20 and 12-lipoxy-genase21 are induced in colonic epithelial cells inresponse to inflammatory mediators like inter-feron y, IL1, and TNFa. The induction ofNGAL synthesis is the first description ofinduc-tion of a lipocalin in colonic epithelial cells. Thismay represent a novel mechanism by whichepithelial cells participate in the regulation ofinflammatory processes as part of the mucosaldefence. The functional significance of theobserved 'neo-expression' ofNGAL in inflamedcolorectal epithelium is very intriguing. It hasbeen shown that the transmigration of neutro-phils across intestinal epithelial monolayersresults in a reduction in the barrier function of

the epithelium, permitting the transepithelialpassage of a variety of tracers.12 As mentioned,NGAL binds a derivative of the bacterialchemotactic formyl-peptide FMIp,29 and couldtherefore serve a function in the colonic epithe-lium as a scavenger of'bacterial products thatwould otherwise traverse the epithelium andfurther recruit both macrophages and neutro-phils (Fig 3). In this context it is interesting tonote that CAIJA (common acute lymphoblas-tic leukaemia antigen), a neutral endopeptidase,has also been shown to be induced in epithelialcells as a result of inflammation.37 Neutralendopeptidase is known to hydrolyse a variety ofphysiologically active peptides includingFMLP.38 This shows that NGAL and neutralendopeptidase might act in concert to inactivatebacterial formyl-peptides. The passage oflipopolysaccharide over the epithelial lining is awell known stimulus for macrophages to pro-duce ILl116 and TNFot,4 which are centralmediators in the inflammatory reaction. Thebinding by NGAL of lipopolysaccharide wouldtherefore be an effective means of downregulat-ing the inflammatory reaction (Fig 3).We conclude that induction of NGAL syn-

thesis is an important cellular response toinflammation in colon epithelium. The faecalcontent of NGAL may therefore prove to be auseful marker for disease activity in inflamma-tory bowel disease. The structure of NGALtogether with its presence in a broad variety oflarge bowel diseases strongly supports its roleas a modulator of the inflammatory response.

No NGAL expression ;in epithelial cells

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420 Nielsen, Borregaard, Bundgaard, Timshel, Sehested, Kjeldsen

The expert technical assistance of Charlotte Horn, Pia L Olsen,Dorte HoIm, and Karen Jensen is greatly appreciated. DrEsther Hage, Department of Pathology, University Hospital,Copenhagen, Denmark is gratefully acknowledged for supply-ing part of the archival material.This work was supported by grants from The Danish Cancer

Society, The Danish Arthritis Association, The Danish MedicalResearch Council, The Lundbeck Fund, Emil C Hertz's Fund,The Novo Fund, Amalie J0rgensen's Fund, Brochner-Mortensen's Fund, Anders Hasselbach's Fund, Ane KathrinePlesners Fund. NB is the recipient of a Neye-ResearchProfessorship.

Part of this work has been published in abstract form (Eur JfClin Invest 1995; 25 (suppl 2): A33).

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