functional andclinical myelomonocyte calprotectin · conformational changes related to calcium...

I Clin Pathol:Mol Pathol 1997;50:1 13-123

Reviews

Functional and clinical aspects of themyelomonocyte protein calprotectin

B Johne, M K Fagerhol, T Lyberg, H Prydz, P Brandtzag, C F Naess-Andresen, I Dale

Calprotectin is a calcium binding heterocom-plex protein consisting of two heavy and onelight chain.' 2 It belongs to the S-100 proteinfamily and is derived predominantly from neu-trophils and monocytes.' This protein isdistributed in myelomonocytic cells, epithelialcells, and keratinocytes and in various tissuesand fluids in the body,' and is a putativeprotective protein.3 Calprotectin and itssubunits appear to have regulatory functions inthe inflammatory process,5 6 and various bio-

Diagnostics R & D, logical functions including antimicrobial7-' andNycomed Pharma AS, antiproliferative'° " activity have been ascribedPO Box 5012 Maj, to the protein. In acute phase inflammatoryN-0301 Oslo, Norway reactions calprotectin is detectable in elevatedB Johne amounts that, in some instances correlate to

elevated levels of neutrophil granulocytes orBlood Bank and other inflammation parameters such as C reac-

Imnunology and tive protein (CRP) or erythrocyte sedimenta-Transfusion Medicine, tion rate (ESR). '`-` Sander et al'5 found poorUllevaal University correlation between calprotectin and CRP,Hospital, N-0407 Oslo, blood leucocytes, and ESR in life threateningNorway infections, thus suggesting that these para-M K Fagerhol meters reflect separate aspects of the inflamma-C F Naess-Andresen tory response.I Dale The clinical relevance of calprotectin meas-Research Forum urements has been described in several diseaseT Lyberg conditions, particularly in inflammatory dis-

eases and certain microbial infections, as wellDepartient of as neoplastic conditions.'6 17 The highest rise inPediatrics calprotectin concentrations can be found inP Brandtzeeg cystic fibrosis, rheumatoid arthritis, Crohn'sBiotechnology Centre disease, ulcerative colitis, and bacterialof Oslo, University of infections."'..4 18 19Oslo, Oslo, Norway Why do we find calprotectin so fascinating?H Prydz There are already numerous markers of

Correspondence to: inflammatory reactions9 2"26 including granulo-Dr Johne cyte markers, so why introduce another?email: berit.johne( Calprotectin is a multipotent biologically activenycomed.telemax.no molecule.' We know that large molecules can

Accepted for publication have separate domains with different biological27 February 1997 functions27 2'8 and that cellular functions (in

Table 1 Calprotectin nomenclature

Nomenclature References *

Calprotectin ( Li) (2 heavy + 1 light chain) Fagerhol,' 2Fagerhol et al"MRP 8/14 (MIF related protein) Odink et al34 Sorg'Cystic fibrosis associated antigen (CFA) Wilson,35 36 Dorin"7

(identity with calprotectin and MRP-8) (Anderson et ap8 39Calgranulins A and B Wilkinson et alPS-iOOa and b (calcium binding proteins) Dorin et al,317 4Freemont et al27

*First publications and/or key references.

macrophages) can be selectively and simulta-neously up or downregulated via different enti-ties on such macromolecules."8 Calcium bind-ing proteins of the S-100 protein family areinvolved in complex intracellular signaltranductions,'7 '9 and it has been suggested thatcalprotectin plays an important role in themetabolism of myeloid cells.30 Furthermore,when calprotectin is external to cells it hasimmunomodulatory functions5' and an im-portant role in neutrophil defence againstmicrobial infections.21 31 32 Antitumour activityhas also been suggested,'" " as well as an innatedefence function.3 Thus, calprotectin appearsto be an important regulatory protein insidethe myeloid cells and extracellularly in inflam-matory reactions. This remarkable spectrum offunctions demonstrated in a recently discov-ered protein prompted an up to date review asa basis for further studies.

NomenclatureCalprotectin is an elusive protein with manynames2; several independent research groupshave called it Li protein, MRP-8/14, calgranu-lin, and cystic fibrosis antigen (table 1).

Calprotectin was first isolated from granulo-cytes as described by Fagerhol et al in 198033and named Li protein. The name calprotectinwas proposed later8 when calcium binding andantimicrobial activity had been documented.Sorg's group5 worked with macrophages andmechanisms of chronic inflammation, and theMIF related proteins MRP-8, MRP-14, andtheir heterocomplex MRP-8/14 were describedin 1987 by Odink.34 A cystic fibrosis associatedantigen was first described by Wilson et al in197335 and further characterised by Wilkinsonet alt0 who proposed the name calgranulin in1988. The identity between the Li protein,cystic fibrosis antigen, and MRP-8 andMRP-14 was established in 1988 by aminoacid and cDNA sequencing, and immunohis-tochemical staining.38 39 Dorin et al37 41 andFreemont et af" further characterised theS-100 structure of these proteins.

Calprotectin is a suitable descriptive namefor this multipotent calcium binding proteinwith protective properties.'

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14ohne, Fagerhol, Lyberg, Prydz, Brandtzceg, Naess-Andresen, et al

Table 2 Physicochemical properties

Molecular weight 36 kDa2Two heavy chains of 14 kDa2One light chain of 8 kDa2Each chain binds two calcium ions42Zinc binding43Heat resistant42Resistant to proteolysis when calcium is present' 2Immunogenic4MoAb defined epitopes

Mac38745'4'27EiO5S 36.4848S 32.2, 8-5C249CF145, CF55746Fl1, F3, A150CP- 1, -2, -551

Strong bond between subunits' 2Forms noncovalent complexes (-di, -tri, and tetramers) in a

calcium dependent manner2352S-100 protein structure and sequence identity'7 375'55Mapped to human chromosome I (and murine 3)41 48 56

The references cited are representative reports or reviews, not a

complete list of publications on each topic.MoAb, monoclonal antibody.

Structure and physicochemicalpropertiesThe physicochemical properties of calprotectin(table 2) have been reviewed thoroughly.' It is a

36 kDa heterotrimeric calcium binding proteinwith two heavy and one light chain non-

covalently linked. 2 42 In the presence ofEDTAit is anionic and migrates in electrophoresis as

an a2 globulin, whereas in the presence ofcalcium it is slightly basic and migrates as a y

globulin. Free heavy and light chains have beendetected only after dissociating treatment,typically by heating in the presence of sodiumdodecyl sulphate (SDS) and 8 M urea followedby SDS polyacrylamide gel electrophoresis or

two dimensional electrophoresis (isodalt). Thepolypeptide chains may be purified by isoelec-tric focusing in the presence of 6 M urea.57 58

Reactive sulphydryl groups are exposed afterdissociation, and unless these are blocked or

alkylated, various heterodimers may beformed.52 59 Addition of calcium may promoteformation of such complexes and even calpro-tectin dimers-that is, double heterotrimers.57 42

Epitope mapping of the protein, by use of a

series of overlapping seven amino acid longsynthetic peptides covering the heavy and lightchains, has shown that rabbits produce antibod-ies against three different linear epitopes on

each chain (Hansen et al, 1995, unpublished).Many monoclonal antibodies against calprotec-tin have been produced (table 2). Epitope map-ping of calprotectin with six murine mono-

clonal antibodies revealed four separateepitopes CJohne and Hansen, 1995, unpub-lished). Binding of calcium causes conforma-tional changes in calprotectin as demonstratedby circular dichroism42 and reactivity withmonoclonal antibodies.5' Furthermore, calciummakes the protein remarkably resistant againstheat and proteolysis.2 42 Its S-100 like proteinstructure has been established' and the geneshave been sequenced and mapped to humanchromosome 1, q I2-q2 1." 48

Distribution in cells and tissuesCalprotectin is found in cells, tissues, andfluids in all parts of the human body (table 3).It is mainly a myelomonocyte and keratinocyte

Table 3 Distribution in cells and tissues

CellsNeutrophil granulocytes33 57 58Monocytes/macrophages9 34 54Epithelial cells20 60-62Keratinocytes63-65Pancreatic cell lines66Tracheal gland cells67

TissuesSkin (epidermis/dermis)62 68Lung'6Gut69Oral mucosa6" 64Cervix mucosa65Body fluids' (see table 6)

Other speciesMouse70Rat'0 71Pig72Sheep73Rabbit74

The references cited are representative reports or reviews, not acomplete list of publications on each topic.

protein. In neutrophils, calprotectin is locatedin the extralysosomal cytosol in concentrationsestimated at 5-15 mg/ml,33 57 thus constitutingabout 5% of total proteins in neutrophil granu-locytes. By immunoelectron microscopy, smallamounts were also found in electron denseparts of myelomonocyte nuclei (Staubli andFagerhol, 1991, unpublished). The calprotec-tin chains are expressed in monocytes and acti-vated macrophages but in decreasing amountswith increasing differentiation of the latter.Calprotectin is variably expressed on thesurface membrane of granulocytes andmonocytes.75 Dendritic cells may be induced toLi expression in certain reactive states. 'Various mucosal squamous epithelia have beenshown to express cytoplasmic calprotectin evenin the normal state.60 Merten and Figarella67reported calprotectin secretion in trachealgland cells, and Fanjul et at6 demonstratedMRP-8 and MRP- 14 in pancreatic cells.Calprotectin is also found in rat, mouse, rabbit,sheep, cattle, and pig.1° 70-74 76 It is thus anabundant ubiquitous molecule.

Biological functionRecent publications have revealed numerousbiological functions of the calprotectinmolecule in vitro and in vivo (table 4). Thestructural identification as an S- 100-likeprotein and its calcium dependent associationto cytoskeleton structures29 suggests intra-cellular signal transduction functions.87Translocation has been shown of phosphoryl-ated calprotectin chains to the membraneduring human neutrophil activation,88 andinhibition of intracellular enzymes importantin cell proliferation (casein kinase II andtopoisomerases).89 It was demonstrated thatthe protein is phosphorylated, and this raisesthe possibility that calprotectin may be a com-peting substrate for the enzyme. The firstdocumentation of antimicrobial activity8 hasbeen confirmed in several papers (table 4), andhas given calprotectin a central role inneutrophil defence.2' 90 91 The abundance anddistinctive properties of calprotectin suggeststhat it plays an important role in neutrophilbiology.32 92 Stimulation of immunoglobulinproduction,6 chemotactic factor activity,83 and

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Functional and clinical aspects of the myelomonocyte protein calprotectin

Table 4 Biologicalfunctions

Intracellular signal transduction (S-i 00 function)27 47 7Calcium dependent association to cytoskeleton structures29 77

Antimicrobial activity8 21 31 78 79Ant Candida albicans activity33 80-82Neutrophil defence mechanism2' 32Stimulation of immunoglobulin production5Chemotactic factor83Neutrophil immobilising factori7Regulatory protein in inflammatory reactions5Marker of myelomonocytic cell differentiation8485Cytotoxic effects:

a) cytotoxic factor in rat peritoneal excudate cells'"b) induction of apoptosis"

Protective action against rheumatoid arthritis (rat model)88


neutrophil immobilising factor activity27 arealso functions in a non-specific defence reper-toire, and related to its properties as a regula-tory protein in inflammatory reactions.'3Calprotectin is a marker for myelomonocyticcell differentiation,84 85 and it has cytokine-likeeffects.'81 Brun et al6 demonstrated protec-tive action of injected calprotectin in a ratmodel of avridin induced rheumatoid arthritis,confirming its regulatory role in inflammatoryreactions. Sorg5 discussed the importance ofthe single protein chains versus the hetero-complex in chronic inflammation.Data by Hahn and Sohnle,9 Clohessy and

Golden,8' 82 and Sohnle et at3 suggest that theantimicrobial action is at least in part the resultof binding to zinc. It has been shown88 that cal-protectin may inhibit metalloproteinases,which may also involve deprivation ofzinc. Thezinc binding site is separate from the calciumbinding sites on MRP-14.43 Thus calprotectinis a large multipotent biologically activemolecule with defined regions or epitopes withdistinct functions. There is, however, much tobe done regarding the investigation of structureeffect relations. Particularly exciting are theconformational changes related to calciumbinding, and its importance for molecularfunctions.

Clinical relevanceThe mere fact that no calprotectin deficiencyhas been found (among more than 5000 indi-viduals tested) may suggest that this protein isof vital importance. The clinical relevance ofcalprotectin in a number of disease conditionshas been suggested by several authors (table5). The first measurements of concentration33in plasma from normal subjects and frompatients with inflammatory, infectious, andmalignant diseases have been followed byextensive documentation with immunohisto-chemical methods, and with immunometricconcentration measurements in various bodyfluids (table 6). In several disorders thedifferences between normal and pathologicallevels are large enough to suggest a diagnosticpotential for calprotectin. 15 17 18 97 105 106 118

Early attention was paid to rheumaticdiseases.'3 .. .. Cystic fibrosis was studiedbefore the protein was identified and character-ised, and several other inflammatory diseaseshave since been studied (table 5). Therelevance of calprotectin in malignant diseaseshas also been pointed out.'6 17 50 123

CALPROTECTIN IN PLASMAIncreased concentration of calprotectin inplasma is found in many types of infectious ororganic diseases.'5 This seems logical in view ofthe large amounts of the protein in myelo-monocytic cells, their active role in defenceagainst infections, and their participation ininflammatory processes and removal of deadcells. We therefore hypothesised that plasmacalprotectin concentrations reflect the turnoverof myelomonocytic cells in the body. Plasmaconcentrations during viral infections rarelyexceed 2 mg/l, while in bacterial infections val-ues above 3 mg/l are seen regularly.

In a series of patients with meningococcalinfections, calprotectin concentrations at hos-pital admission were between 10 and 120 mg/lin those with fulminant septicaemia (table 7).As calprotectin is preformed and ready to bereleased from activated circulating leucocytes,its concentration in plasma can increase muchmore rapidly in response to bacteraemia andendotoxaemia than acute phase proteins(CRP) synthesised in the liver. The latter has adoubling time of three to four hours, so it maytake from six hours to more than a day for theplasma concentration to reach more than theupper reference limit. If a rapid test isdeveloped for use in primary health care,plasma calprotectin determinations may con-tribute to the distinction between viral andbacterial infections and proper use of antibiot-ics. Sander et all'5 concluded that "low ornormal Li levels argue strongly against bacte-rial infection, while elevated LI levels discrimi-nate poorly between bacterial and non-infectious inflammatory or malignant disease."

Upregulation of S-100 protein CP-10 byendotoxin via distinct pathways has been dem-onstrated in murine cells in vitro.126 It may becalculated that if most of the calprotectin inneutrophil granulocytes (about 5 pg/cell) in theblood of a healthy human individual (contain-ing about 4 x 109 neutrophils per liter) isreleased in to the plasma, the concentration isexpected to increase from 0.5 to about 20 mg/l.

Table 5 Clinical relevance of calprotectin reported invarious disease conditions

Blood donors (plasma reference levels)"3Rheumatoid arthritis'2 13 22 94Sjogrens syndrome91Intraocular inflammatory conditions95Systemic lupus erythematosus96Cystic fibrosis40 97Acute and chronic lung disease98Lung carcinoma (squamous cells)'6Soft tissue tumor marker (non-specificity)89 100Colorectal cancer'7 101 102Crohn's disease'9 103 104Ulcerative colitis'9 105 106Gastrointestinal mucosal inflammation'07 108

Urinary stone'09Oral inflammatory mucosal disease84CNS inflammatory disease (multiple sclerosis and acute

encephalitis) l10Secondary CNS infections in HIV infected patients'8HIV infected patients7 lHaematological patients50Febrile conditions infectious and non-infectious18Acute myocardial infarction"2Surgery' 14-116Apheresis' 1'


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6Johne, Fagerhol, Lyberg, Prydz, Brandtzeeg, Naess-Andresen, et al

Table 6 Calprotectin concentrations in various body fluids

Bodyfluid References Calprotectin concentrations*

Serum/plasma Sander et al'5

Muller et al"9Dale"3

Berntzen et al'2"14

Haga et al6Semb et al"4

Garred et al"'

Lugering 103 106

Golden et al'

Ivanov et alP

Cerebrospinal fluid

Oral fluids

Urine

Faeces

Synovial fluid

Empyema fluid (supernatant)

Dunlop et al"

Brun et al'

Cuida et al'2'

Muller et al"'

Holt et al'20

Roseth et al'9

Roseth et al"Gilbert et al'02

Meling et al'09

Berntzen et al'4

Santanagopalan et al'9

Infectious diseasesNormal 0.1-0.6Viral 0.1-1.4Bacterial 0.6-11.0

HIV infection (25-75 percentiles) 1.2-9.4Reference intervals

Females 0.09-0.53Males 0.12-0.66

Rheumatic diseaseNormal range 0.8-0.91Rheumatoid arthritis 1-46Osteoarthritis 0.5-0.8Juvenile rheumatoid arthritis 2-24Systemic lupus erythematosus, mean 3.6

Cardiopulmonary bypass operationsPreoperative 0.3Postoperative 5.2±1.3

Major surgeryPreoperative baseline 0.5-0.9Postoperative (4h) 7-15

Crohn's diseaseActive 17±6 (MRP 8/14)Inactive 5±2 (MRP 8/14)

Cystic fibrosis childrenControls 0.3-1.6 (median 0.7)Cystic fibrosis 0.4-26 (median 1.8)

HaematologyHealthy blood donors 0.2Leukaemia 0.4-13.3 (MRP 8/14)

Normal range 0-37 ,sg/lHIV positive with infections 30-350,g/lSj0gren's syndrome

Stimulated whole saliva 23.6Healthy subjects

Parotis saliva 3.2Stimulated whole saliva 22.0Mucosal transudate 40.9

HIV infected with or without oral candidiasisParotis 0.06-0.41

Infants and children (range)Controls 24 pg/l (5-650)Cystitis 182 sg/l (18-992)Pyelonephritis 1000 sg/ml (360-7000)

Median (range)Healthy subjects 2 (0.5-8)Controls 10.5 (1.1-80)Crohn's disease 43 (8-2000)Ulcerative colitis 19.5 (2.4-866.4)

Colorectal cancer 50.0 (4.5-950)Healthy controls, range 0-9Colorectal cancers (mean (SD))

Right side 55.1 (58.9)Left side 79.3 (58.2)

Mean (95% confidence intervals)Normal baseline 4.9 (1.5-15.6)NSAID treated 9.0 (6-27)

Median (range)Rheumatoid arthritis 18 (2-375)Osteoarthritis 0.9 (0.2-2)

Not quantitated. Calprotectin functionalassay for C albicans growth inhibition

* Concentrations in cerebrospinal fluid and urine given as pg/l, all others given as mg/I.

Such values are found regularly in plasma fromcitrated blood stored for 14-21 days.

Increased plasma calprotectin concentra-tions are found regularly in HIV infected indi-viduals, and a strong further increase inresponse to zidovudine treatment seems topredict a favourable prognosis. "' Curiously,increased plasma calprotectin is associatedwith a poor chance of survival in patients withalcoholic liver disease irrespective of the degreeof parenchymal liver damage.'27During surgery, complement activation can

be demonstrated by increased concentrationsofthe terminal complement complex or C3b inplasma. Simultaneously, granulocyte lysosomalproteins (myeloperoxidase and lactoferrin) andcalprotectin increase, but the latter remainselevated many hours longer than the former,even after the complement activation signals

have returned to baseline."16 128 129 Coronarysequestration of both granulocytes and calpro-tectin was demonstrated in the early reper-fusion period after cardiac surgery and coldcardioplegic arrest."4

Recently, Arvesen et al"2 found that plasmacalprotectin concentrations are increased inpatients with coronary artery disease. Evenpatients with unstable angina or small (non-Q)myocardial infarctions had mean values aboutthree times the upper reference limit. An inter-esting possibility is that the elevations mayreflect an increased (inherited or acquired?)myelomonocytic response to even minorstimuli that in the long run may predispose toatherosclerosis.Plasma calprotectin levels are increased in

active rheumatic diseases; Berntzen et al'2-14found 1-46 mg/l in rheumatoid arthritis

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Table 7 Inflammation markers in meningococcal infections

Calprotectin CRP Leukocytes LPS IL-6

Clinical presentation mg/i range mg/i range x E911 range ngll range ,ugll range

Fulminant sepsis* 13.8 10.5-119.9 85 19-225 7.1 2.2-33.3 2800 210-170000 550 55-2400Meningitis* 6.8 2.9-51.0 137 24-328 22.6 3.8-30.9 <3 <3-210 0 0-297Mildsystmenin* 6.7 1.6-14.3 75 16-162 14.6 9.5-35.0 45 <3-600 3 0-102Reference valuet 0.7 0.3-1.6 S 10 <3 <0.05

*Fulminant sepsis plasma (n= 13) measured 12 h or less after onset of symptoms. Meningitis plasma (n=20) measured 24 h or less after onset. Mild systemic menin-gococcal (syst menin) disease plasma (n=6) measured at variable time intervals after onset of symptoms. Median value and range is given for each parameter.t Reference values are from Golden et al" for calprotectin, Waage et al'24 for lipopolysaccharide (LPS) and interleukin (IL)-6, and Hjortdal et all2" for C reactive pro-tein (CRP). See also Brandtzeeg and Kierulf."3

patients and 2-24 mg/l in juvenile rheumatoidarthritis, while Haga et ar6 reported a meanvalue of 3.6 mg/l in systemic lupus erythemato-sus patients compared with 1.05 mg/l inmatched controls. In both rheumatoid arthritisand systemic lupus erythematosus, plasma cal-protectin values appear to be an objectiveparameter for the assessment of disease activityand response to treatment. Furthermore, bothplasma and synovial fluid calprotectin clearlydistinguish rheumatoid from osteoarthritis.'4Plasma calprotectin concentrations are in-

creased in endogenous posterior uveitis.95Patients may also have raised antineutrophiliccytoplasmatic antibodies (ANCA), but theANCA titres did not correlate with calprotectinlevels. The authors suggested the latter may bea sensitive indicator of disease activity forendogenous posterior uveitis. There seems tobe a similar situation for cystic fibrosis.97

In a series of patients with newly diagnosedpulmonary cancers,'23 increased plasma calpro-tectin was found in 81 %, while other diseaseindicating parameters lagged behind: CRP,45%; orosomucoid, 48%; haptoglobulin, 59%;a, antitrypsin, 31 %; alanine aminotransferase,5%; aspartate aminotransferase, 0%; yglutamyl transferase, 27%; alkaline phos-phatase, 10%. Strikingly, about 15% of thepatients had calprotectin concentrations (andother markers) close to or below the upper ref-erence limit despite advanced, invasive disease.This raises the possibility that some tumoursmay release substances capable of inhibitingthe emigration or activation ofmyelomonocyticcells. At least such cells would be expected toparticipate in the removal of dead tumour cells.

CALPROTECTIN IN CEREBROSPINAL FLUIDLimited data have been published, but calpro-tectin levels in CSF seem to distinguishbetween HIV encephalitis and opportunisticinfections in the central nervous system in HIVinfected patients.'8

CALPROTECTIN IN ORAL FLUIDSConcentrations between 3 and 40 mg/l havebeen measured in different oral secretions(table 6). Muller et al'l found lower calprotec-tin concentrations in HIV infected patientswho suffered from oral candidiasis comparedwith all HIV infected subjects, and with healthycontrols.

CALPROTECTIN IN URINEIncreased calprotectin is found in patients withurinary tract infections, particularly in patientswith renal involvement.'20 Calprotectin

measurement has been suggested as a urinarymarker of asthma.'30

CALPROTECTIN AND GASTROINTESTINAL DISEASESR0seth et al'9 developed a method for thedetermination of calprotectin in stool as analternative to al antitrypsin for the evaluationof disease activity in inflammatory boweldisease (IBD). It was, however, soon realisedthat increased faecal calprotectin is a marker ofdiseases of the gastrointestinal tract, includinggastric cancer, colorectal adenoma or cancer,Crohn's disease, and ulcerative colitis. 910' 105The method depends on the preparation of asimple buffer extract of a small spot sample ofstool, and quantitation of calprotectin byenzyme linked immunosorbent assay (ELISA).Only a fraction of the total calprotectin contentin the stool sample is brought into solution, andmost of it is found in high molecular size com-plexes (Roseth and Fagerhol, 1992, unpub-lished).

In recently diagnosed colorectal cancers,stool calprotectin concentrations above anupper reference limit of 10 mg/l were found inmore than 90% of the patients.'9 In this studythe value decreased below 10 mg/l aftersuccessful radical resection. No significant dif-ferences in calprotectin were found in cancersof Duke's stages A and B compared with C andD, which suggests that a positive calprotectintest may be found even in early cancers. This issupported by the finding of increased concen-trations in more than 60% of patients withcolorectal adenoma irrespective of size andlocation.'0' Again, calprotectin concentrationsreturned to normal after removal of theadenomas.'0' Although calprotectin is also syn-thesised by squamous epithelial cells, it is notfound in epithelial cells of the bowel nor incolorectal cancer cells. The numbers ofneutrophils, however, are increased signifi-cantly in the peritumoral stroma in coloniccarcinomas.'3' The working hypothesis is thatdifferent types of pathological changes in thegastrointestinal tract cause increased perme-ability of the mucosa leading to increasedmigration of granulocytes and monocytestowards chemotactic substances in the gutlumen. An important factor for the high sensi-tivity of the faecal calprotectin test is the factthat this protein is remarkably resistant to pro-teolysis in the presence of calcium.2 This mayexplain why the test has been found positive inmore than 90% of patients with gastriccancer. 19

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It is notable that faecal calprotectin sheddingmay be caused by drugs'08 or other inflamma-tory reactions in the gastrointestinal system.Thus, extensive clinical studies are beingconducted in this area, particularly aimed atconfirming a negative predictive value forcolorectal cancer. 7 Haemoglobin or faecaloccult blood are used extensively as markers inscreening for colorectal cancer. Calprotectin isa less variable parameter than haemoglobin infaecal samples from patients with colorectalcancers,'02 and although both parameters areelevated significantly, they do not correlate.Thus the mechanism of luminal calprotectinentry appears to be both different from and lesserratic than bleeding.'02

In patients with active IBD, faecal calprotec-tin concentrations above 100 mg/l are regularlyfound,'0' and a diagnosis of IBD should not bemade in a symptomatic patient with a faecalcalprotectin level below 10 mg/l. Highly signifi-cant correlations were found between this testand the IBD disease activity determined byendoscopy, histology or excretion of "'indiumlabelled autologous granulocytes."' 105 The lat-ter method has been regarded as the "goldstandard" but its use is limited by complexity,cost, and exposure of the patient to irradiation.The relatively simple faecal calprotectin test isan attractive alternative as it can be repeated atany time and, because of the stability of calpro-tectin, samples can be mailed by the patient tothe laboratory. Treatment response in patientswith Crohn's disease can be monitored by fae-cal calprotectin measurements.

CONCLUSIONThe clinical relevance of calprotectin seems tobe related to its physiological functions duringhomeostasis as well as in pathogenesis. Thesefunctions are intracellular as well as extracellu-lar. Disease modulation during rheumatoidarthritis and possible antitumour effects of cal-protectin may result from specific mechanismsother than antimicrobial activity. Calprotectinis a non-specific marker for activation ofgranulocytes and mononuclear phagocytes. Itis released from these cells and is thus expectedto be found in secretory and excretoryproducts in different parts of the body.

Possible clinical applicationsThe diagnostic value of calprotectin has beenemphasised in several publications (tables 5and 6) that recommend calprotectin as aninflammatory disease marker. Extensive andsystematic clinical documentation is still lack-ing, however, some larger studies are ongoing.RIA"3 and ELISA' have been established asimmunoassays for calprotectin measurement inbody fluids, and are also applied to stoolsamples."'' Two commercial ELISA assaysexist, with an analytical sensitivity at the ng/mllevel. In the NycoCard (Nycomed, Oslo, Nor-way) immunoassay format'25 132 a prototypequantitative rapid test for calprotectin has beenestablished with an analytical sensitivity ofapproximately 0.05 gg/ml. Documentation willincrease as commercial calprotectin tests be-come easier and more widely available. Immu-

nohistochemical staining for calprotectin maybe a diagnostic tool in inflammatory skindiseases, malignant conditions, and a variety ofother diseases.3 133 Several commercial andnon-commercial monoclonal antibodies areavailable (table 2).Monitoring of disease activity and therapy

may be relevant in inflammatory diseases suchas rheumatoid arthritis, cystic fibrosis, Crohn'sdisease, asthma,94 97 130 and others (tables 5 and6).

Therapeutic use of calprotectin (or some ofits structural elements) is a fascinating thoughtwith its protective biological properties.Lehrer32 calls it "an internal ointment thatrestricts the growth of C albicans on nearbyskin." Could it be used as an externalointment? In terms of therapy could themolecule be used in rheumatic, inflammatory,infectious or malignant diseases?

Unpublished dataSYNTHESIS AND RELEASE OF CALPROTECTIN FROMHUMAN MONOCYTES IN VITROA series of experiments were performed imme-diately after the discovery of calprotectin, togather data concerning the mechanisms in-volved in the release of calprotectin frommonocytes. These data have not previouslybeen published, nor have similar data beenpublished by others. We have therefore in-cluded them in the present review.

Materials and methodsHuman monocytes were isolated from healthyfasting donors as described previously'34 35 andcultured in RPMI 1640 (Gibco-Biocult, Pais-ley, Scotland) containing 20% inactivated fetalcalf serum (Flow, Irvine, Scotland). The finalcell preparations consisted of more than 95%monocytes demonstrated by differential count-ing, phagocytosis and staining for non-specificesterase.'36 The presence of calprotectin wasdetermined by single radial diffusion or ELISAafter incubation periods of up to 46 hours. Thesynthesis of calprotectin was studied by addi-tion of 35S-methionine to the medium, andradiolabelled calprotectin was determined byan immunoaffinity column with immobilised,monospecific rabbit anti-calprotectin antibod-ies.The following compounds were added to

monocyte cultures: immune complexes formedby incubating equivalent amounts of humanserum albumin or transferrin (Sigma, St Louis,Missouri, USA) and corresponding antibodies(Dakopatts, Copenhagen, Denmark); aggre-gated human IgG (Gammaglobulin, Kabi,Stockholm, Sweden) prepared by heating a 1%solution in 0.15 M NaCl at 63°C for 12minutes, and precipitation with Na2SO4 (67 g/l);cross-linked human immunoglobulins preparedby reacting IgG (10 g/l) with 0.5% glutaralde-hyde in 0.1 M acetate buffer, pH 4.0 for onehour at room temperature, followed by dialysisagainst distilled water and phosphate bufferedsaline, pH 7.35; concanavalin A (ConA) andwheat germ agglutinin (WGA) (Sigma); phyto-haemaglutinin (PHA) (Wellcome, Beckenham,Kent, UK); ionophores A 23187 and nigericin

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Table 8 Release of calprotectin from human monocytes after 16 hours in vitro culture

Concentration Serum * No. of CalprotectinStimulant ,uglml 20% cultures releaset (ng

LectinsConA 25 + 14 < 25ConA 25 - 2 98PHA 25 + 8 49PHA 25 - 2 130WGA 25 + 4 46WGA 25 - 2 1050

Antigen/antibody complexesHSA/anti-HSA 10t + 8 665HSA/anti-HSA 10t - 2 265Transferrin/antitransferrin 2t + 6 440Heat aggregated IgG (IgG-H) 700 + 2 15Glutaraldehyde aggregated IgG

(IgG-G) 700 + 2 25HSA/anti-HSA + IgG-H 10t + 700 + 2 48HSA/anti-HSA + IgG-H 10* + 250 + 2 52HSA/anti-HSA + IgG-G 10t + 700 + 2 37HSA/anti-HSA + IgG-G 10* + 250 + 2 49

IonophoresA 23187 0,25 + 14 165A 23187 0,25 - 6 319Nigericin 2,5 + 2 > 1000

Endotoxin 25 + 12 136Phorbol esterTPA 1 + 2 2238

* Fetal calf serum inactivated at 56'C for 30 min.t The release from unstimulated control cells was < 25 nglml.* Amount of antigen present in the immune complex added.HSA, human serum albumin.

Table 9 Release of lysozyme and /3 glucuronidase from stimulated human monocytesi;vitro

Lysozyme % of fi glucuronidase % ofunstimulated unstimulated control

Stimulant control (SEM) Cultures (n) (SEM) Cultures (i

Immune complexes 695 (189) 12 610 (79) 8Endotoxin 138 (45) 10 107 (4) 8A 23187 435 (251) 12 198 (68) 10Lectins 92 (8) 16 97 (21) 14Nigericin 257 2 356 2TPA 664 (114) 10 425 (70) 10

* The final concentrations of stimulants were as given in table 8.

O TPA, 0.5 pg/mla IC, 10 pg/mIA LPS, 25 pg/mI

(Lilly Research Corporation, Indianapolis, Indi-ana, USA); endotoxins E coli 011 :B4 and E coli

1ml) 055:B5 (Difco, Detroit, Michigan, USA); ac-- tinomycin D (Serva, Heidelberg, Germany);

1 2-O-tetradecanoylphorbol- 1 3-acetate (TPA)(PL Biochemicals, Milwakee, Wisconsin, USA);cycloheximide, cytochalasin B, and prostaglan-din El (PGE,) (Sigma).

Lactate dehydrogenase, lysozyme, and P glu-coronidase activities were determined in cul-ture supernatants and cell homogenates.'37-139

ResultsFreshly prepared monocytes contained2.5 ± 0.7 pg calprotectin per cell, and afterincubation for 9-10 hours the content was2.3 ± 0.2 pg/cell.

Various agents known to interact with andactivate'40 monocytes were added to monocytecultures. None of these, except TPA at 1 ptg/mlcaused cytotoxicity (increased trypan blueuptake) within the time period studied. Asshown in table 8, very small amounts of calpro-tectin were released from monocytes culturedfor 16 hours in the presence of lectins and 20%inactivated fetal calf serum. In the absence ofserum slightly higher amounts (but less than

*n 1% of total cellular calprotectin content) werereleased after ConA or PHA stimulation, whileWGA caused a noticeably higher release. Norelease of lactate dehydrogenase or : glucuro-

en) nidase, and no increased lysozyme release wasseen in lectin stimulated cultures (table 9).Immune complexes led to a marked release ofcalprotectin over two to three hours (fig 1), andabout 8% of the calculated total cellularcalprotectin was released. This release was twoto threefold higher in the presence of fetal calfserum (table 8). Native serum was 25% moreactive than inactivated serum, and nativeserum alone promoted the release of calprotec-tin in the absence ofimmune complexes. Addi-tion of immune complexes induced a markedrelease of both ,B glucuronidase and lysozyme(table 9), but essentially no release of lactatedehydrogenase.

Phorbol ester (TPA) at 500 ng/ml caused aquite rapid and marked release of calprotectin(about 7% of total cellular content), but nosigns of cytotoxicity in three hours (fig 1).Release of the total intracellular calprotectinwas seen when a cytotoxic dose of TPA (1 gg/ml) was applied for 14 hours (table 8). Thedivalent ionophore A23 187 caused a moderaterelease of calprotectin, and the monovalentionophore nigericin a substantial release (table8). Both ionophores increased moderately therelease of ,B glucuronidase and lysozyme (table9).Endotoxin induced a moderate but distinct

release of calprotectin (table 8), but no signifi-cant increase in lysozyme or f glucuronidasesecretion (table 9). A very small amount (lessthan 2% of total cellular content) was released

1 during the first three hours (fig 1).200 To learn more about the mechanism of

calprotectin release, addition of several othercompounds were tested. Prostaglandin E,

he (10 jM) did not influence the release ofcalprotectin during incubation with TPA. The

1000

100

C

0c)

0.

(o

2010

0 50 100 150Minutes

Figure 1 Time course for release of calprotectin from human monocytes in vitro.TPA, 12-0-tetradecanoylphorbol-13-acetate; LPS, endotoxin; IC, immune complexes. Tfinal concentrations in pglml of the added substances are indicated.

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10ohne, Fagerhol, Lyberg, Prydz, Brandtzag, Naess-Andresen, et al

Table 10 Release of calprotectin, lysozyme, andfi glucuronidase from stimulated humanmonocytes in vitro, with potentially interfering substances added

Stimulant* Addinonst Calprotectint Lysozyme fi glucuronidaset

Immune complexes Dexamethasone 101 (7) 84 (7) 87 (35)Promethazine 98 131 134Indomethacin 81 (12) 100 (5) 115 (8)

Endotoxin Dexamethasone 44 (6) 99 (15) 141Promethazine 248 (70) 1250 597 (260)

A 23187 Dexamethasone 122 93 (7) 18Promethazine 311 452 426Indomethacin 196 (12)

Lectins Dexamethasone 95 (25) 101 (18) 58 (15)Indomethacin 230

* The final concentrations of stimulants were as given in table 8.t Final concentrations: dexamethasone, 10 g/ml; promethazine and indomethacin; 0.1 mM.t Each value is the mean of 2-8 cultures, and calculated as % of stimulated control (SEM).

prostaglandin synthesis inhibitors indometh-acin and acetylsalicylic acid had no effect onthe release caused by immune complexes orlectins (table 10). Release induced by A23187was enchanced twofold by indomethacin butnot by acetylsalicylic acid. As indomethacin hasother effects besides blocking prostaglandinsynthesis,14' our tentative conclusion is thatproducts of the prostaglandin pathway are notinvolved in calprotectin secretion. The antihis-tamine promethazine enhanced the release ofcalprotectin induced by endotoxin, A23187 orTPA (data not shown), whereas the releaseinduced by immune complexes was unchanged(table 10). Similarly, both lysozyme and 1 glu-curonidase release was enchanced by additionof promethazine to A23187 or endotoxins, butnot to immune complex stimulated monocytes(table 9). Dexamethasone inhibited calprotec-tin release induced by endotoxins but not thatinduced by A23187, lectins or immune com-plexes. This drug had no effect on the lysozymerelease, but the release of 1 glucuronidaseinduced by A23187 and lectins was inhibited.Cytochalasin B (10 jtg/ml), which interfereswith actin polymerisation, had no effect on cal-protectin release induced by endotoxin, but atwofold enhancing effect in the presence ofPHA.Dexamethasone (10 jg/ml), dibutyryl cAMP

(1 mM), cAMP (1 mM), verapamil (25 jg/ml), acetylsalicylic acid (0.1 mM), and purifiedprotein derivative of Bacillus Calmette-Guerin(PPD) had no effect of calprotectin releasefrom unstimulated cells.

DiscussionThe release of calprotectin from isolatedmonocytes can result from general cytotoxicity.If sufficiently high doses are used-for exam-ple, TPA at 1 ,ug/ml, the total calprotectin poolmay be released. However, partial release wasobserved without concomitant lactate dehy-drogenase release or trypan blue uptake. Thissuggests that either calprotectin release is avery sensitive indicator of cytotoxicity or therelease is caused by other mechanisms.There was no definite evidence for involve-

ment of the prostaglandin system in calprotec-tin release, and the fraction released duringthree to four hours never exceeded 8% of thetotal cellular pool.The agents that promoted lysosomal enzyme

release most (immune complexes, TPA, iono-phores) also increased the calprotectin release,

although not exactly in the same proportion. Acorrelation was observed between lysozymeand calprotectin release. The effects of dexa-methasone served to distinguish these pro-cesses in that it reduced calprotectin releasewhile leaving lysozyme and 1 glucuronidaserelease unchanged in response to endotoxin. Inthe presence of lectins or A23187 the oppositewas found: dexamethasone reduced 13 glucuro-nidase release whereas calprotectin and lys-ozyme release were unchanged. It is thereforelikely that calprotectin release is different fromthe lysozyme and lysosomal release processes.Clearly, calprotectin release can be caused by anumber of membrane perturbing agents, andcalprotectin is either derived from the plasmamembrane or from an intracellular compart-ment by some special release process.'42

Anti-calprotectin antibodies added tohuman monocytes in culture induced the syn-thesis of the protein component ofthromboplastin,'40 which suggests that calpro-tectin is at least in part located in the plasmamembrane, as observed by Dale et ar' andGuignard et al.88

ConclusionsCalprotectin is fascinating because it holdsunrevealed secrets. We know that calprotectinis an important granulocyte marker and a multi-functional regulatory protein in inflammatoryprocesses. There is more work to do in order tounderstand fully basic mechanisms such asquaternary structure, complex assembly, mo-lecular functions, and biological effect mecha-nisms.Much has been reported regarding the clini-

cal relevance and diagnostic potential ofcalprotectin and its subunits, and larger clinicalstudies are continuing. Further work maygather speed as commercial kits for calprotec-tin measurement become more readily avail-able. Interesting therapeutic concepts may befound for calprotectin, as the relation of thestructure and function of the molecule are bet-ter understood.

In the meantime we will do as Lehrer"recommended in 1993: "Stay tuned. Calpro-tectin, whatever it is, could be interesting."

1 Fagerhol MK, Anderson KB, Naess-Andresen CF,Brandtzxg P, Dale I. Calprotectin (the LI leukocyteorotein). In: Smith VL, Dedman JR, eds. Stimulus responsecoupling: the role of intracellular calcium-binding proteins. BocaRaton: CRC Press Inc, 1990:187-210.

2 Fagerhol MK. Nomenclature for proteins: is calprotectin aproper name for the elusive myelomonocytic protein? Jf ClinPathol: Mol Pathol 1996;49:M74-9.

3 Brandtzwg P, Dale I, Gabrielsen TO. The leukocyte proteinLi (calprotectin): usefulness as an immunohistochemicalmarker antigen and putative biological function. Histopa-thology 1992;21:191-6.

4 Brandtzmg P, Gabrielsen T-0, Dale I, Muller F, SteinbakkM, Fagerhol MK. The leukocyte protein LI (calprotectin):a putative nonspecific defence factor at epithelial surfaces.Adv Exp Med Biol 1995;371:201-6.

5 Sorg C. The calcium binding proteins MRP8 and MRP14in acute and chronic inflammation. Behringer Inst Mitt1992;91:126-37.

6 Brun JG, Ulvestad E, Fagerhol MK, Jonsson R. Effects ofhuman calprotectin (LI) on in vitro immunoglobulin syn-thesis. Scand J Immunol 1994;40:675-80.

7 McNamara MP, Weissner JH, Collins-Lech C, Hahn JH,Sohnle PG. Neutrophil death as a defence mechanismagainst Candida albicans infections. Lancet 1988;ii: 1163-5.

8 Steinbakk M, Naess-Andresen C-F, Lingaas E, Dale I,Brandtzaeg P, Fagerhol MK. Antimicrobial actions ofcalcium binding leucocyte Li protein, calprotectin. Lancet1990;336:763-5.

120 on D



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9 Hahn BL, Sohnle PG. Resistance of zinc-supplementedCandida albicans cells to the growth inhibitory effect ofcalprotectin. J7 Infect Dis 1995;171:1289-94.

10 Yui S, Mikami M, Yamazaki M. Purification and characteri-zation of the cytotoxic factor in rat peritoneal exudate cells:its identification as the calcium binding protein complex,calprotectin. J Leukocyte Biol 1995;58:307-16.

11 Yui S, Mikami M, Yamazaki M. Induction of apoptotic celldeath in mouse lymphoma and human leukemia cell linesby a calcium-binding protein complex, calprotectin,derived from inflammatory peritoneal exudate cells. 7 Leu-kocyte Biol 1995;58:650-8.

12 Berntzen HB, Endresen GK, Fagerhol MK, Spiechowicz J,Mowinckel P. Calprotectin (the Li protein) during surgeryin patients with rheumatoid arthritis. Scand J Clin LabInvest 1991;51:643-50.

13 Berntzen HB, Fagerhol MK, 0stensen M, Mowinckel P,Hryeraal HM. The Li protein as a new indicator of inflam-matory activity in patients with juvenile rheumatoid arthri-tis.JRheumatol 1991;18:1338.

14 Berntzen HB, Olmez U, Fagerhol MK, Munthe E. The leu-kocyte protein LI in plasma and synovial fluid frompatients with rheumatoid arthritis and osteoarthritis. ScandJ Rheumatol 199 1;20:74-82.

15 Sander J, Fagerhol MK, Bakken JS, Dale I. Plasma levels ofthe leukocyte LI protein in febrile conditions: relation toaetiology, number of leukocytes in blood, blood sedimenta-tion reaction and C-reactive protein. Scand Jf Clin LabInvest 1984;44:357-62.

16 Dale I, Brandtzscg P. Expression of the epithelial Li antigenas immunohistochemical marker of squamous cell carci-noma of the lung. Histopathology 1989;14:493-502.

17 R0seth AG, Kristinsson J, Fagerhol MK, Schonsby H,Aadland E, Nygaard K, et al. Faecal calprotectin: a noveltest for the diagnosis of colorectal cancer? Scand J Gastro-enterol 1993;28:1073-6.

18 Dunlop 0, Bruun JN, Myrvang B, Fagerhol MK. Calprotec-tin in cerebrospinal fluid of the HIV infected: a diagnosticmarker of opportunistic central nervous system infection?ScandJtInfect. Dis 199 1;23:687-9.

19 Roseth AG, Fagerhol MK, Aadland E, Sch0nsby H. Assess-ment of the neutrophil dominating protein calprotectin infeces. A methodologic study. Scandj3 Gastroenterol 1992;27:793-8.

20 Brandtz2eg P, Dale I, Fagerhol MK. Distribution of aformalin-resistant myelomonocytic antigen (L1) in humantissues. 1. Comparison with other leukocyte markers bypaired immunofluorescence and immunoenzyme staining.Am J Clin Pathol 1987;87:681-99.

21 Miyasaki KT. The Neutrophil: mechanisms of controllingperiodontal bacteria. J Periodontol 1991 ;62:761-74.

22 Brun JG, Haga H-J, Boe E, Kallay I, Lekven C, BerntzenHB, et al. Calprotectin in patients with rheumatoidarthritis: relation to clinical and laboratory variables of dis-ease activity. J Rheumatol 1992;19:859-62.

23 Brandtzag P, Kierulf P. Endotoxin and meningococcemia.Intravascular inflammation induced by native endotoxin inman. In: Ryan JL, Morrison DC, eds. Bacterial endotoxiclipopolysaccharides. Vol II. Immunopharmacology and patho-physiology. Boca Raton: CRC Press, 1992:327-46.

24 Hammer HB, Kvien TK, Glennas A, Melby K. A longitudi-nal study of calprotectin as an inflammatory marker inpatients with reactive arthritis. Clin Exp Rheumatol1995;13:59-64.

25 Kjeldsen-Kragh J, Mellbye OJ, Haugen M, Mollnes TE,Hammer HB, Sioud M, et al. Changes in laboratoryvariables in rheumatoid arthritis patients during a trial offasting and one-year vegetarian diet. Scand J Rheumatol1995;24:85-93.

26 Soderquist B, Sundqvist KG, Jones I, Holmberg H,Vikerfors T Interleukin-6, C-reactive protein, lactoferrinand white blood cell count in patients with S aureus septi-cemia. ScandlInfect Dis 1995;27:375-80.

27 Freemont P, Hogg N, Edgeworth J. Sequence identity.Nature 1989;339:516.

28 Johne B, Bryn K, Gaudernack G, Myhrvold V, M0rland B.Activation of mononuclear phagocyte functions by struc-turally different bacterial endotoxins. In: Fossum S, ed.Advances in experimental medicine. Vol 237. Histophysiologyof the immune system: The life history, organization, and inter-actions of its cellpopulations. New York: Plenum Press, 1989:793-7.

29 Kelly SE, Hunter JA, Jones DB, Clark BR, Fleming S. Mor-phological evidence for calcium-dependent association ofcalgranulin with the epidermal cytoskeleton in inflamma-tory dermatoses. BrJ Dermatol 1991;124:403-9.

30 Bengis-Garber C, Gruener N. Calcium-binding myeloidprotein (P8,P14) is phosphorylated in fMet-Leu-Phe-stimulated neutrophils. J Leukocyte Biol 1993;54:114-18.

31 Miyasaki KT, Bodeau AL, Murthy ARK, Lehrer RI. In vitroantimicrobial activity of the human neutrophil cytosolicS-100 protein complex, calprotectin, against Capnocy-tophaga sputigena. J Dent Res 1993;72:517-23.

32 Lehrer RI. Holocrine secretion of calprotectin: a neutrophil-mediated defence against Candida albicans? J Lab ClinMed 1993;121:193-4.

33 Fagerhol MK, Dale I, Anderson I. Release and quantitationof a leucocyte derived protein (L1). Scand J Haematol1980;24:393-8.

34 Odink K, Cerletti N, Bruggen J, Clerc RG, Tarcsay L,Zwadlo G, et al. Two calcium-binding proteins in infiltratemacrophages of rheumatoid arthritis. Nature 1987;330-80-2.

35 Wilson GB, Jahn TL, Fonseca JR. Demonstration of serumprotein differences in cystic fibrosis by isoelectic focussingin thin-layer polyacrylamide gels. Clin Chim Acta 1973;49:79-85.

36 Wilson GB, Fudenberg HH, Jahn TL. Studies on cysticfibrosis using isoelectric focusing. I. An assay for detectionof cystic fibrosis homozygotes and heterozygote carriersfrom serum. Pediatr Res 1975;9:635-40.

37 Dorin JR, Novak M, Hill R, Brock D, Secher D, vanHeyningen V. A clue to the basic defect in cystic fibrosisfrom cloning the CF antigen gene. Nature 1987;326:614-7.

38 Anderson KB, Sletten K, Berntzen HB, Dale I, BrandtzaegP, Jellum E, et al. The leukocyte LI protein: identity withthe cyctic fibrosis antigen and the calcium-binding MRP-8and MRP-14 macrophage components. Scand J Immunol1988;28:241-5.

39 Anderson KB, Sletten K. Leukocyte Li protein and thecystic fibrosis antigen. Nature 1988;332:688.

40 Wilkinson MM, Busuttil A, Hayward C, Brock DJ, DorinJR, van Heyningen V. Expression pattern of two relatedcystic fibrosis-associated calcium-binding protein in nor-mal and abnormal tissues. J Cell Sci 1988;91:221-30.

41 Dorin JR, Emslie E, van Heyningen V. Related calcium-binding proteins map to the same subregion of chromo-some lq and to an extended region of synteny on mousechromosome 3. Genomics 1990;8:420-6.

42 Naess-Andresen CF, Egelandsdal B, Fagerhol MK. Cal-cium binding and concomitant changes in the structureand heat stability of calprotectin (Li protein). J Clin Pathol:Mol Pathol 1995;48:M278-84.

43 Raftery MJ, Harrison CA, Jones A, Geczy CL. Isolation ofthe murine S100 protein MRPi4 (14 kDa migration-inhibitory-factor-related protein) from activated spleencells: characterization of post-translational modification ofzinc binding. BiochemJ1996;316:285-93.

44 Dale I, Fagerhol MK, Naesgaard I. Purification and partialcharacterization of a highly immunogenic human leucocyteprotein, the Li antigen. Eur3 Biochem 1983;134:1-6.

45 Brandtzxg P, Jones DB, Flavell DJ, Fagerhol MK. Mac 387antibody and detection of formalin resistant myelomono-cytic Li antigen.J Clin Pathol 1988;41:963-70.

46 Kelly SE, Jones DB, Flemming S. Calgranulin expression ininflammatory dermatoses. J Pathol 1989;159: 17-21.

47 Guignard F, Mauel J, Markert M. The monoclonal antibodyMac 387 recognizes three S100 proteins in humanneutrophils. Immunol Cell Biol 1996;74:105-7.

48 Lagasse E, Clerc RG. Cloning and expression of two humangenes encoding calcium-binding proteins that are regulatedduring myeloid differenciation. Mol Cell Biol 1988;8:2402-10.

49 Delabie J, DeWolf-Peeters C, van den Oord JJ, Desmet VJ.Differential expression of the calcium-binding proteinsMRP8 and MRP14 in granulomatous conditions. Animmunohistochemical study. Clin Exp Immunol 1990;81:123-6.

50 Ivanov GE, Misuno NI, Ivanov VE. Enzyme linkedimmunosorbent assay for human MRP-8/MRP-14 pro-teins and their quantitation in plasma of hematologicalpatients. Immunol Lett 1996;49:7-13.

51 Johne B. Epitope mapping by surface plasmon resonance inthe BIAcore. In: Morris G, ed. Methods in molecular biology.Vol 66. Epitope mapping protocols. Totowa: Humana Press,1996.

52 Teigelkamp S, Bhardwaj RS, Roth J, Meinardus-Hager G,Karas M, Sorg C. Calcium-dependent complex assemblyof the myeloic differentiation proteins MRP-8 andMRP-14.J Biol Chem 1991;266:13462-7.

53 Madsen P, Rasmussen HH, Leffers H, Honore B, DejgaardK, Olsen E, et al. Molecular cloning, occurrence, andexpression of a novel partially secreted protein "psoriasin"that is highly up-regulated in psoriatic skin. J Invest Derma-tol 1991;97:701-12.

54 Hessian PA, Edgeworth J, Hogg N. MRP-8 and MRP-14,two abundant Ca"+-binding proteins of neutrophils andmonocytes. J Leukocyte Biol 1993;53:197-204.

55 Hessian PA, Wilkinson L, Hogg N. The S100 family proteinMRP-14 (S100A9) has homology with the contact domainof high molecular weight kininogen. FEBS Lett 1995;371:271-5.

56 van Heyningen V, Hayward C, Fletcher J, McAuley C. Tis-sue localization and chromosomal assignment of a serumprotein that tracks the cystic fibrosis gene. Nature1985;315:513-5.

57 Berntzen HB, Fagerhol MK. Li, a major granulocyteprotein: antigenic properties of its subunits. Scand J ClinLab Invest 1988;48:647-52.

58 Berntzen HB, Fagerhol MK. LI, a major granulocyteprotein; isolation of high quantities of its subunits. ScandJClin Lab Invest 1990;50:769-74.

59 Longbottom D, Sallenave JM, van Heyningen V. Subunitstructure of calgranulins A and B obtained from sputum,plasma, granulocytes and cultured epithelial cells. BiochimBiophysActa 1992;1120:215-22.

60 Brandtzxg P, Dale I, Fagerhol MK. Distribution of a forma-lin resistant myelomonocytic antigen (LI) in humantissues. 2. Normal and aberrant occurrence in various epi-thelia. Am J Clin Pathol 1987b;87:700-7.

61 van Heyningen V, Dorin J. Possible role for two calcium-binding proteins of the S-100 family, co-expressed ingranulocytes and certain epithelia. Adv Exp Med Biol 1990;269:139-43.

62 Saintigny G, Schmidt R, Shroot B, Juhlin L, Reichert U,Michel S. Differential expression of calgranulin A and B in

121 on D



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p.50.3.113 on 1 June 1997. Dow

nloaded from

http://mp.bmj.com/

12ohne,Fagerhol, Lyberg, Prydz, Brandtzceg, Naess-Andresen, et al

various epithelial cell lines and reconstructed epidermis. JInvest Dermatol 1992;99:639-44.

63 Eversole LR, Miyasaki KT, Christensen RE. The distribu-tion of the antimicrobial protein, calprotectin, in normaloral keratinocytes. Arch Oral Biol 1992;37:963-8.

64 Eversole LR, Miyasaki KT, Christensen RE. Keratinocyteexpression of calprotectin in oral inflammatory mucosaldiseases. Jf Oral Pathol Med 1993;22:303-7.

65 Coleman N, Stanley MA. Expression of the myelomono-cytic antigens CD36 and LI by keratinocytes in squamousintraepithelial lesions of the cervix. Hum Pathol 1994;25:73-9.

66 Fanjul M, Renaud W, Merten M, Guycrotte 0, Hollande E,Figarella C. Presence of MRP8 and MRP14 in pancreaticcell lines: differential expression and localization inCFPAC-1 cells. Am JPhysiol: Cell Physiol 1995;37:C1241-51.

67 Merten MD, Figarella C. Constitutive hypersecretion andinsensitivity to neurotransmitters by cystic fibrosis trachealgland cells. Am J3 Physiol 1993;264:L93-9.

68 Gabrielsen T-O, Dale I, Brandtzag P, Hoel PS, FagerholMK, Larsen TE, et al. Epidermal and dermal distributionof a myelomonocytic antigen (LI) shared by epithelial cellsin various inflammatory skin diseases.3Am Acad Dermatol1986;15:173-9.

69 Bjerke K, Halstensen TS, Jahnsen F, Pulford. K, BrandtzaegP. Distribution of macrophages and granulocytes express-ing LI protein (calprotectin) in human Peyer's patchescompared with normal ileal lamina propria and mesentericlymph nodes. Gut 1993;34:1357-63.

70 Nacken W, Manitz MP, Sorg C. Molecular characterisationof the genomic locus of the mouse MRP8 gene. BiochimBiophysActa 1996;1315:1-5.

71 Naughton PJ, Clohessy PA, Grant G, Pusztai A, Golden B.Faecal calprotectin: non-invasive marker of gastrointestinalinflammation in salmonella infected rats. Biochem Soc Trans1996;24:308S.

72 Dell'Angelica EC, Schleicher CH, Santome JA. Primarystructure and binding properties of calgranulin C, a novelS100-like calcium-binding protein from pig granulocytes. .7Biol Chem 1994;269:28929-36.

73 Belenko M, Chanana AD, Joel DD, Fagerhol MK, Janoff.Tracing the leukocyte marker protein in lung fluids andlung-draining lymph nodes during endotoxemia in sheep.Am Rev Resp Dis 1986;133:866-74.

74 Yang Z, Deveer MJ, Gardiner EE, Devenish RJ, Handley CJ,Underwood JR, et al. Rabbit polymorphonuclear neu-trophils form S-35-labeled S-sulfo-calgranulin C whenincubated with inorganic [S-35]sulfate. _7 Biol Chem 1996;271:19802-9.

75 Dale I, Brandtzaeg P, Fagerhol MK, Scott H. Distribution ofa new myelomonocytic antigen (LI) in human periferalblood leukocytes. Am J Clin Pathol 1985;84:24-34.

76 Dianoux A-C, Stasia M-J, Garin J, Gagnon J, Vignais PV.The 23-kilodalton protein, a substrate of protein kinase C,in bovine neutrophil cytosol is a member of the S 100 fam-ily. Biochemistry 1992;31:5898-905.

77 Clark BR, Kelly SE, Flemming S. Calgranulin expressionand association with the keratinocyte cytoskeleton. J Pathol1990;160:25-30.

78 Edgeworth JD, Abiose A, Jones BR. An immunohistochemi-cal analysis of onchocercal nodules: evidence for aninteraction between macrophage MRP8/MRP14 and adultOnchocerca volvulus. Clin Exp Immunol 1993;92:84-92.

79 Santhanagopalan V, Hahn BL, Dunn BE, Weissner JH,Sohnle PG. Antimicrobial activity of calprotectin isolatedfrom human empyema fluid supernatants. Clin ImmunolImmunopathol 1995;76:285-90.

80 Murthy AR, Lehrer RI, Harwig SS, Miyasaki KT. In vitrocandidiastic properties of the human neutrophil calprotec-tin complex. J Immunol 1993;151:6291 -301.

81 Clohessy PA, Golden BE. Calprotectin mediated zinc chela-tion as a biostatic mechanism in host defence. Scand JImmunol 1995;42:551-6.

82 Clohessy PA, Golden BE. The mechanism of calprotectin'scandidastic activity appears to involve zinc chelation.Biochem Soc Trans 1996;24:309S.

83 Kocher M, Kenny PA, Farram E, Majid KBA, FinlayjonesJJ, Geczy CL. Functional chemotactic factors CP-10 andMRP-14 are abundant in murine abscesses. Infect Immun1996;64:1342-50.

84 Zwadlo G, Bruggen J, Gerhards G, Schlegel R, Sorg C. Twocalcium-binding proteins associated with specific stages ofmyeloid cell differentiation are expressed by subsets ofmacrophages in inflammatory tissues. Clin Exp Immunol1988;72:510-5.

85 Murao S, Nakanishi M, Matsumoto S, Ueda N, HubermanE. Induction of cell differentiation in human HL-60promyelocytic leukemia cells: Quantitation of a myeloidspecific antigen, MRP-8/MRP-14 protein complex. Cellbiology-a laboratory handbook. Vol 1. London: AcademicPress, 1994:207-12.

86 Brun JG, Haaland G, Haga HJ, Fagerhol MK, Jonsson R.Effects of calprotectin in avridine-induced arthritis.APMIS 1995;163:233-40.

87 Kligman D, Hilt DC. The S100 protein family. TIBS 1988;13:437-43.

88 Guignard F, Mauel J, Markert M. Phosphorylation ofmyeloid-related proteins MRP-14 and MRP-8 during neu-trophil activation. Eur3rBiochem 1996;241:265-71.

89 Murao S, Collart FR, Huberman E. A protein containingthe cystic fibrosis antigen is an inhibitor of protein kinases._FBiol Chem 1989;254:8356-60.

90 Sohnle PG, Collins-Lech C, Wiessner JH. Antimicrobialactivity of an abundant calcium-binding protein in thecytoplasm of human neutrophils. _J Infect Dis 1991;163:187-92.

91 Brun JG, Cuida M, Jacobsen H, Kloster R, Johannesen AC,Hoyeraal HM, et al. Sj0gren's syndrome in inflammatoryrheumatic diseases: Analysis of the leukocyte proteincalprotectin in plasma and saliva. ScandJ Rheumatol 1994;23:114-18.

92 Levy 0. Antibiotic proteins of polymorphonuclear leuko-cytes. Eur3r Haematol 1996;56:263-77.

93 Sohnle P G, Hahn B L, Santanagopalan V. Inhibition ofCandida albicans growth by calprotectin in the absence ofdirect contact with the organisms. J Infect Dis 1996;174:1369-72.

94 Berntzen HB, Munthe E, Fagerhol MK. A longitudinalstudy of the leukocyte protein LI as an indicator of diseaseactivity in patients with rheumatoid arthritis. J Rheumatol1989;16:1416-20.

95 Olson JA, Forrester M, Clohessy PA, Golden BE, Herriot R,Forrester JV. Calprotectin is raised in endogenousposteri-our uveitis. Occular Immunol Inflamm 1996;4:91-8.

96 Haga HJ, Brun JG, Berntzen HB, Cervera R, KhamashtaM, Huges GRV. Calprotectin in patients with systemiclupus erythematosus: Relation to clinical and laboratoryparameters of disease activity. Lupus 1993;2:47-50.

97 Golden BE, Clohessy PA, Russell G, Fagerhol MK. Calpro-tectin as a marker of inflammation in cystic fibrosis. ArchDis Child 1996;74:136-9.

98 Stockley RA, Dale I, Hill SL, Fagerhol MK. Relationship ofneutrophil cytoplasmic protein (Li) to acute and chroniclung disease. Scandj7 Clin Lab Invest 1984;44:629-34.

99 Wick MR, Mills SE, Swanson PE. Expression of "myelo-monocytic" antigens in mesotheliomas and adenocarcino-mas involving the serosal surfaces. Am J Clin Pathol1990;94:18-26.

100 Lofthus B, Loh LC, Curran B, Henry K, Leader M.Mac387: its non-specificity as a tumor marker or marker ofhistiocytes. Histopathology 1991;19:251-5.

101 Roseth AG, Kristinsson J, Schonsby H, Nygaard K,Raknerud N, Fagerhol MK, et al. Faecal calprotectin in aconsecutive series of outpatients referred for colonoscopy,with special emphasis on inflammatory bowel disease andcolorectal neoplasia. Digestion. [In press.]

102 Gilbert JA, Ahlquist AA, Mahoney DW, Zinsmeister AR,Rubin J, Ellefson RD. Fecal marker variability in colorectalcancer: calprotectin versus hemoglobin. Scand _' Gastroen-terol 1996;31:1001-5.

103 Lugering N, Stoll R, Kucharzik T, Schmid KW, RohlmannG, Burmeister G, et al. Immunohistochemical distributionand serum levels of the Ca2"-binding proteins MRP8,MRPP14 and their heterodimeric form MRP8/14 in Crohn'sdisease. Digestion 1995;56:406-14.

104 Schmid KW, Lugering N, Stoll R, Brinkbaumer P, WindeG, Damschke W, et al. Immunohistochemical demonstra-tion of the calcium-binding proteins MRP8 and MRP14and their heterodimer (27E1 0 antigen) in Crohn's disease.Hum Pathol 1995;26:334-7.

105 Roseth AG, Aadland E, Jahnsen J, Raknerud N. Assess-ment of disease activity in ulcerative colitis by faecalcalprotectin, a novel granulocyte marker protein. Digestion1997;58: 176-80.

106 Lugering N, Stoll R, Schmid KW, Kucharzik T, Stein H,Burmeister G. The myeloic related protein MRP8/14(27E10 antigen)-usefulness as a potential marker for dis-ease cativity in ulcerative colitis and putative biologicalfunction. Eur3 Clin Invest 1995;25:659-64.

107 Foster R, Bjarnason IB, Roseth AG, Fagerhol MK, PetersTJ. Alcohol misuse causes a reversible inflammatory enter-opathy with increased intestinal permeability. Alcohol Alco-holism 1995;30:C5.2.

108 Meling T, Aabakken L, Roseth A, Osnes M. Faecalcalprotectin-shedding after short term treatment with non-steroidal anti inflammatory drugs. Scand J Gastroenterol1996;31:339-44.

109 Umekawa T, Kurite T. Calprotectin-like protein is relatedto soluble organic matrix in calcium oxalate urinary stone.Biochem. Mol Biol Int 1994;34:309-13.

110 Ulvestad E, Williams K, Mork S, Antel J, Nyland H. Phe-notypic differences between human monocytes/macrophages and microglial cells studied in situ and invitro. J Neuropathol Exp Neurol 1994;53:492-501.

111 Muller F, Froland SS, Brandtzaeg P, Fagerhol MK. Oralcandidiasis is associated with low levels of parotidcalprotectin in individuals with infection due to humanimmunodeficiency virus. Clin Infect Dis 1993;16:301-2.

112 Arvesen K, Kontny F, Toss H, Wallentin L, Roseth A,Sundset A, et al. Calprotectin: a novel plasma marker ofgranulocyte activation in acute coronary syndrome [ab-stract]. EurHeartJ7 1996;17:429.

113 Dale I. Plasma levels of the calcium-binding Li leukocyteprotein: standardization of blood collection and evaluationof reference intervals in healthy controls. Scand J Clin LabInvest 1990;50:837-41.

114 Semb AG, Gabrielsen TO, Halstensen TS, Fagerhol MK,BrandtzEeg P, Vaage J. Cardiac surgery and distribution ofthe leukocyte Li protein-calprotectin. . Cardiothorac Surg1991;5:363-7.

115 Garred P, Fosse E, Fagerhol MK, Videm V, Mollnes TE.Calprotectin and complement activation during majoroperations with or without cardiopulmonary bypass. AnnThorac Surg 1993;55:694-9.

116 Fosse E, Moen 0, Johnson E, Semb G, Brockmeier V,Mollnes TE, et al. Reduced complement and granulocyte

122 on D



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p.50.3.113 on 1 June 1997. Dow

nloaded from

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activation with heparin-coated cardiopulmonary bypass.Ann Thorac Surg 1994;58:472-7.

117 Hetland G, Berntzen HB, Fagerhoel MK. Levels ofcalprotectin (leukocyte LI protein) during apheresis. ScandClin Lab Invest 1992;52:479-82.

118 Lugering N, Stoll R, Kucharsik T, Burmeister G, Sorg C,Domschke W. Serum 27E10 antigen: a new potentialmarker for staging HIV disease. Clin Exp Immunol1995;101:249-53.

119 Muller F, Fr0land S, Aukrust P, Fagerhol MK. Elevatedserum calprotectin levels in HIV-infected patients: the cal-protectin response during ZDV treatment is associatedwith clinical events.3JAcq Immun Defic Syndr 1994;7:931-9.

120 Holt J, Fagerhol MK, Dale I. Quantitation of a leukocyteprotein (LI) in urine. Acta Paediatr Scand 1983;72:615-16.

121 Cuida M, Brun JG, Tynning T, Jonsson R. Calprotectinlevels in oral fluids: the importance of collection site. EurJOral Sci 1995;103:8-10.

122 Sohnle PG, Collins-Lech C, Wiessner JH. The zinc-reversible antimicrobial activity of neutrophil lysates andabscess fluid supernatants. 7 Infect Dis 1991;164: 137-42.

123 Fagerhol MK, Dale I, Boye N, Fjeld N, Urianstad M.Plasma Li protein levels: a marker for early pulmonarycancer [abstract]. Clin Res 1985;33:617A.

124 Waage A, Brandtzag P, Halstensen A, Kierulf P, Espevik T.The complex pattern of cytokines in serum from patientswith meningococcal septic shock. Exp Med 1989;169:333-8.

125 Hjortdal P, Landaas S, Urdal P, Steinbakk M, Fuglerud P,Nygaard B. C-reactive protein: a new rapid assay for man-aging infectious disease in primary health care. PrimHealth Care 1991;3:3-10.

126 Hu S-P, Harrison C, Xu K, Cornish CJ, Geczy CL. Induc-tion of the chemotactic S100 protein, CP-1 0, in monocyte/macrophages by lipopolysaccharide. Blood 1996;87:3919-28.

127 Homann C, Garred P, Graudal N, Hasselquist P,Christiansen M, Fagerhol MK, et al. Plasma calprotectin: Anew prognostic marker of survival in alchohol-induced cir-rosis. Hepatology 1995;21:979-85.

128 Pekna M, Borowiec J, Fagerhol MK, Venge P, Thelin S.Biocompatibility of heparin-coated curcuits used in cardi-opulmonary bypass. Scand 7 Thorac Cardiovasc Surg 1994;28:5-11.

129 Moen 0, Fosse E, Braten J, Andersson C, Fagerhol MK,Hogasen K, et al. Roller and centrifugal pumps comparedin vitro with regard to haemolysis, granulocyte andcomplement activation. Perfusion 1994;9: 109- 17.

130 Shute J, Fagerhol MK, Parmar J, Holgate S, Howarth P.Calprotectin, a urinary marker of disease activity in severeasthma [abstract]. BALR Summer Meeting 1996.

131 Svennevig J-L, Lunde OC, Holter J. In situ analysis of theinflammatory cell infiltrates in colon carcinomas and in thenormal colon wall. Acta Path Microbiol Immunol Scand1982;90:131-7.

132 Johne B, Hansen K, Mork E, Holtlund J. Colloidal goldconjugated monoclonal antibodies, studied in the BIAcorebiosensor and in the NycoCard immunoassay format. JImmunol Methods 1995;183:167-74.

133 Rugtveit J, Scott H, Halstensen TS, Norstein J, BrandtzaegP. Expression of the Li antigen (calprotectin) by tissuemacrophages reflects recent recruitment from peripheralblood rather than upregulation of local synthesis: Implica-tions for rejection diagnosis in formalin-fixed kidney speci-mens. Jf Pathol 1996; 180:194-9.

134 Prydz H, Allison AC. Tissue thromboplastin activity ofisolated human monocytes. Thrombos Haemostas 1978;39:582-91.

135 Prydz H, Lyberg T, Deteix P, Allison AC. In vitro stimula-tion of tissue thromboplastin (factor III) activity in humanmonocytes by immune complexes and lectins. Thromb Res1979;15:465-74.

136 Yam LT, Li CY, Crosby WH. Cytochemical identificationof monocytes and granulocytes. Am J Clin Path 1971;55:283-90.

137 Kirkeby K, Prydz H. Lactic dehydrogenase activity inpleural and peritoneal effusions. Scand J Clin Lab Invest1959;11:185-9.

138 Gordon S, Todd J, Cohn ZA. In vitro synthesis of lysozymeby mononuclear phagocytes. J Exp Med 1974;139:1228-48.

139 Gianetto R, deDuve C. Tissue fractionation studies. 4.Comparative study of the binding of acid phosphatase,beta-glucoronidase and cathepsin by rat liver particles. Bio-chemJ_ 1955;59:433-8.

140 Prydz H, Lyberg T. Effect of some drugs on thromboplas-tin (factor III) activity of human monocytes in vitro.Biochem Pharm 1980;29:9-14.

141 van Calker D, Mhller M, Hamprecht B. Indomethacin insubmicromolar concentrations inhibits cyclic AMP-dependent protein kinase. Nature 1978;276:841-2.

142 Roth J, Goebler M, Wrocklage V, Bos C, Sorg C. Expres-sion of the calcium binding proteins MRP8 and MRP 14 inmonocytes is regulated by a calcium-induced suppressormechanism. Biochem J 1994;301:655-60.

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