Reviews Mol Path: First Published As 10.1136/Mp.50.3.113 on 1 June 1997

I Clin Pathol:Mol Pathol 1997;50:1 13-123 113 Reviews Mol Path: first published as 10.1136/mp.50.3.113 on 1 June 1997. Downloaded from

Functional and clinical aspects of the myelomonocyte 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- macrophages) can be selectively and simulta- plex protein consisting of two heavy and one neously up or downregulated via different enti- light chain.' 2 It belongs to the S-100 protein ties on such macromolecules."8 Calcium bind- family and is derived predominantly from neu- ing proteins of the S-100 protein family are trophils and monocytes.' This protein is involved in complex intracellular signal distributed in myelomonocytic cells, epithelial tranductions,'7 '9 and it has been suggested that cells, and keratinocytes and in various tissues calprotectin plays an important role in the and fluids in the body,' and is a putative metabolism of myeloid cells.30 Furthermore, protective protein.3 Calprotectin and its when calprotectin is external to subunits appear to have regulatory functions in cells it has the inflammatory process,5 6 and various bio- immunomodulatory functions5' and an important role in neutrophil defence against Diagnostics R & D, logical functions including antimicrobial7-' and microbial infections.21 31 32 Antitumour activity Nycomed Pharma AS, antiproliferative'° " activity have been ascribed has also been " PO Box 5012 Maj, to the protein. In acute phase inflammatory suggested,'" as well as an innate N-0301 Oslo, Norway reactions calprotectin is detectable in elevated defence function.3 Thus, calprotectin appears B Johne amounts that, in some instances correlate to to be an important regulatory protein inside elevated levels of neutrophil granulocytes or the myeloid cells and extracellularly in inflam- Blood Bank and other inflammation parameters such as C reac- matory reactions. This remarkable spectrum of http://mp.bmj.com/ Imnunology and tive protein (CRP) or erythrocyte sedimenta- functions demonstrated in a recently discov- Transfusion Medicine, tion rate (ESR). '`-` Sander et al'5 found poor ered protein prompted an up to date review as Ullevaal University correlation between calprotectin and CRP, a basis for further studies. Hospital, N-0407 Oslo, blood leucocytes, and ESR in life threatening Norway infections, thus suggesting that these para- M K Fagerhol meters reflect separate aspects of the inflamma- Nomenclature C F Naess-Andresen tory response. Calprotectin is an elusive protein with many I Dale on September 29, 2021 by guest. Protected copyright. The clinical relevance of calprotectin meas- names2; several independent research groups Research Forum urements has been described in several disease have called it Li protein, MRP-8/14, calgranu- T Lyberg conditions, particularly in inflammatory dis- lin, and cystic fibrosis antigen (table 1). eases and certain microbial infections, as well Calprotectin was first isolated from granulo- 17 Departient of as neoplastic conditions.'6 The highest rise in cytes as described by Fagerhol et al in 198033 Pediatrics calprotectin can P Brandtzeeg concentrations be found in and named Li protein. The name calprotectin cystic fibrosis, rheumatoid arthritis, Crohn's was proposed later8 when calcium binding and Biotechnology Centre disease, ulcerative colitis, and bacterial antimicrobial activity had been documented. of Oslo, University of infections."'..4 18 19 Sorg's group5 worked with macrophages and Oslo, Oslo, Norway Why do we find calprotectin so fascinating? H Prydz There are already numerous markers of mechanisms of chronic inflammation, and the MIF related proteins MRP-8, MRP-14, and Correspondence to: inflammatory reactions9 2"26 including granulo- their heterocomplex MRP-8/14 were described Dr Johne cyte markers, so why introduce another? in 1987 by Odink.34 A cystic fibrosis associated email: berit.johne( Calprotectin is a multipotent biologically active nycomed.telemax.no molecule.' We know that large molecules can antigen was first described by Wilson et al in 197335 and further characterised by Wilkinson Accepted for publication have separate domains with different biological 27 February 1997 et alt0 who proposed the name calgranulin in functions27 2'8 and that cellular functions (in 1988. The identity between the Li protein, Table 1 Calprotectin nomenclature cystic fibrosis antigen, and MRP-8 and MRP-14 was established in 1988 by amino Nomenclature References* acid and cDNA sequencing, and immunohis- Calprotectin ( Li) (2 heavy + 1 light chain) Fagerhol,' 2Fagerhol et al" tochemical staining.38 39 Dorin et al37 41 and MRP 8/14 ( MIF related protein) Odink et al34 Sorg' Freemont et further characterised the Cystic fibrosis associated antigen (CFA) Wilson,35 36 Dorin"7 af" (identity with calprotectin and MRP-8) (Anderson et ap8 39 S-100 structure of these proteins. Calgranulins A and B Wilkinson et alP Calprotectin is a suitable descriptive name S-iOOa and b (calcium binding proteins) Dorin et al,317 4Freemont et al27 for this multipotent calcium binding protein *First publications and/or key references. with protective properties.' 11414ohne, Fagerhol, Lyberg, Prydz, Brandtzceg, Naess-Andresen, et al

Table 2 Physicochemical properties Table 3 Distribution in cells and tissues

Molecular weight 36 kDa2 Cells Two heavy chains of 14 kDa2 Neutrophil granulocytes33 57 58 One light chain of 8 kDa2 Monocytes/macrophages9 34 54 Each chain binds two calcium ions42 Epithelial cells20 60-62 Mol Path: first published as 10.1136/mp.50.3.113 on 1 June 1997. Downloaded from Zinc binding43 Keratinocytes63-65 Heat resistant42 Pancreatic cell lines66 Resistant to proteolysis when calcium is present' 2 Tracheal gland cells67 Immunogenic4 Tissues MoAb defined epitopes Skin (epidermis/dermis)62 68 Mac38745'4' Lung'6 27EiO5 Gut69 S 36.4848 Oral mucosa6" 64 S 32.2, 8-5C249 Cervix mucosa65 CF145, CF55746 Body fluids' (see table 6) Fl1, F3, A150 Other species CP- 1, -2, -551 Mouse70 Strong bond between subunits' 2 Rat'0 71 Forms noncovalent complexes (-di, -tri, and tetramers) in a Pig72 calcium dependent manner23 52 Sheep73 S-100 protein structure and sequence identity'75'5537 Rabbit74 Mapped to human chromosome I (and murine 3)41 48 56 The references cited are representative reports or reviews, not a The references cited are representative reports or reviews, not a complete list of publications on each topic. complete list of publications on each topic. MoAb, monoclonal antibody. protein. In neutrophils, calprotectin is located Structure and physicochemical in the extralysosomal cytosol in concentrations properties estimated at 5-15 mg/ml,33 57 thus constituting The physicochemical properties of calprotectin about 5% of total proteins in neutrophil granu- (table 2) have been reviewed thoroughly.' It is a locytes. By immunoelectron microscopy, small 36 kDa heterotrimeric calcium binding protein amounts were also found in electron dense with two heavy and one light chain non- parts of myelomonocyte nuclei (Staubli and covalently linked. 2 42 In the presence of EDTA Fagerhol, 1991, unpublished). The calprotec- it is anionic and migrates in electrophoresis as tin chains are expressed in monocytes and acti- an a2 globulin, whereas in the presence of vated macrophages but in decreasing amounts calcium it is slightly basic and migrates as a y with increasing differentiation of the latter. globulin. Free heavy and light chains have been Calprotectin is variably expressed on the detected only after dissociating treatment, surface membrane of granulocytes and typically by heating in the presence of sodium monocytes.75 Dendritic cells may be induced to dodecyl sulphate (SDS) and 8 M urea followed Li expression in certain reactive states.' by SDS polyacrylamide gel electrophoresis or Various mucosal squamous epithelia have been http://mp.bmj.com/ two dimensional electrophoresis (isodalt). The shown to express cytoplasmic calprotectin even polypeptide chains may be purified by isoelec- in the normal state.60 Merten and Figarella67 tric focusing in the presence of 6 M urea.57 58 reported calprotectin secretion in tracheal Reactive sulphydryl groups are exposed after gland cells, and Fanjul et at6 demonstrated dissociation, and unless these are blocked or MRP-8 and MRP-14 in pancreatic cells. alkylated, various heterodimers may be Calprotectin is also found in rat, mouse, rabbit, 70-74 formed.52 59 Addition of calcium may promote sheep, cattle, and pig.1° 76 It is thus an on September 29, 2021 by guest. Protected copyright. formation of such complexes and even calpro- abundant ubiquitous molecule. tectin dimers-that is, double heterotrimers.57 42 Epitope mapping of the protein, by use of a Biological function series of overlapping seven amino acid long Recent publications have revealed numerous synthetic peptides covering the heavy and light biological functions of the calprotectin chains, has shown that rabbits produce antibod- molecule in vitro and in vivo (table 4). The ies against three different linear epitopes on structural identification as an S-100-like each chain (Hansen et al, 1995, unpublished). protein and its calcium dependent association Many monoclonal antibodies against calprotec- to cytoskeleton structures29 suggests intra- tin have been produced (table 2). Epitope map- cellular signal transduction functions.87 ping of calprotectin with six murine mono- Translocation has been shown of phosphoryl- clonal antibodies revealed four separate ated calprotectin chains to the membrane epitopes CJohne and Hansen, 1995, unpub- during human neutrophil activation,88 and lished). Binding of calcium causes conforma- inhibition of intracellular enzymes important tional changes in calprotectin as demonstrated in cell proliferation (casein kinase II and by circular dichroism42 and reactivity with topoisomerases).89 It was demonstrated that monoclonal antibodies.5' Furthermore, calcium the protein is phosphorylated, and this raises makes the protein remarkably resistant against the possibility that calprotectin may be a com- heat and proteolysis.2 42 Its S-100 like protein peting substrate for the enzyme. The first structure has been established' and the genes documentation of antimicrobial activity8 has have been sequenced and mapped to human been confirmed in several papers (table 4), and chromosome 1, q I 2-q2 1." 48 has given calprotectin a central role in neutrophil defence.2' 90 91 The abundance and Distribution in cells and tissues distinctive properties of calprotectin suggests Calprotectin is found in cells, tissues, and that it plays an important role in neutrophil fluids in all parts of the human body (table 3). biology.32 92 Stimulation of immunoglobulin It is mainly a myelomonocyte and keratinocyte production,6 chemotactic factor activity,83 and Functional and clinical aspects of the myelomonocyte protein calprotectin 115

Table 4 Biologicalfunctions CALPROTECTIN IN PLASMA Increased concentration of calprotectin in Intracellular signal transduction (S-i 00 function)27 47 7 Calcium dependent association to cytoskeleton structures29 77 plasma is found in many types of infectious or

Antimicrobial activity8 21 31 78 79 organic diseases.'5 This seems logical in view of Mol Path: first published as 10.1136/mp.50.3.113 on 1 June 1997. Downloaded from Ant Candida albicans activity33 80-82 the large amounts of the protein in myelo- Neutrophil defence mechanism2' 32 Stimulation of immunoglobulin production5 monocytic cells, their active role in defence Chemotactic factor83 against infections, and their participation in Neutrophil immobilising factori7 inflammatory processes and removal of dead Regulatory protein in inflammatory reactions5 Marker of myelomonocytic cell differentiation8485 cells. We therefore hypothesised that plasma Cytotoxic effects: calprotectin concentrations reflect the turnover a) cytotoxic factor in rat peritoneal excudate cells'" b) induction of apoptosis" of myelomonocytic cells in the body. Plasma Protective action against rheumatoid arthritis (rat model)88 concentrations during viral infections rarely exceed 2 mg/l, while in bacterial infections val- The references cited are representative reports or reviews, not a ues above 3 mg/l are seen regularly. complete list of publications on each topic. In a series of patients with meningococcal neutrophil immobilising factor activity27 are infections, calprotectin concentrations at hos- also functions in a non-specific defence reper- pital admission were between 10 and 120 mg/l toire, and related to its properties as a regula- in those with fulminant septicaemia (table 7). tory protein in inflammatory reactions.'3 As calprotectin is preformed and ready to be Calprotectin is a marker for myelomonocytic released from activated circulating leucocytes, cell differentiation,84 85 and it has cytokine-like its concentration in plasma can increase much effects.'81 Brun et al6 demonstrated protec- more rapidly in response to bacteraemia and tive action of injected calprotectin in a rat endotoxaemia than acute phase proteins model ofavridin induced rheumatoid arthritis, (CRP) synthesised in the liver. The latter has a confirming its regulatory role in inflammatory doubling time of three to four hours, so it may reactions. Sorg5 discussed the importance of take from six hours to more than a day for the the single protein chains versus the hetero- plasma concentration to reach more than the complex in chronic inflammation. upper reference limit. If a rapid test is Data by Hahn and Sohnle,9 Clohessy and developed for use in primary health care, Golden,8' 82 and Sohnle et at3 suggest that the plasma calprotectin determinations may con- antimicrobial action is at least in part the result tribute to the distinction between viral and ofbinding to zinc. It has been shown88 that cal- bacterial infections and proper use of antibiot- protectin may inhibit metalloproteinases, ics. Sander et all'5 concluded that "low or which may also involve deprivation ofzinc. The normal Li levels argue strongly against bacte- zinc binding site is separate from the calcium rial infection, while elevated LI levels discrimi-

binding sites on MRP-14.43 Thus calprotectin nate poorly between bacterial and non- http://mp.bmj.com/ is a large multipotent biologically active infectious inflammatory or malignant disease." molecule with defined regions or epitopes with Upregulation of S-100 protein CP-10 by distinct functions. There is, however, much to endotoxin via distinct pathways has been dem- be done regarding the investigation of structure onstrated in murine cells in vitro.126 It may be effect relations. Particularly exciting are the calculated that if most of the calprotectin in conformational changes related to calcium neutrophil granulocytes (about 5 pg/cell) in the

binding, and its importance for molecular blood of a healthy human individual (contain- on September 29, 2021 by guest. Protected copyright. functions. ing about 4 x 109 neutrophils per liter) is released in to the plasma, the concentration is Clinical relevance expected to increase from 0.5 to about 20 mg/l. The mere fact that no calprotectin deficiency has been found (among more than 5000 indi- Table 5 Clinical relevance ofcalprotectin reported in viduals tested) may suggest that this protein is various disease conditions of vital importance. The clinical relevance of calprotectin in a number of disease conditions Blood donors (plasma reference levels)"3 Rheumatoid arthritis'2 13 22 94 has been suggested by several authors (table Sjogrens syndrome91 5). The first measurements of concentration33 Intraocular inflammatory conditions95 in plasma from normal subjects and from Systemic lupus erythematosus96 Cystic fibrosis40 97 patients with inflammatory, infectious, and Acute and chronic lung disease98 malignant diseases have been followed by Lung carcinoma (squamous cells)'6 Soft tissue tumor marker (non-specificity)89 100 extensive documentation with immunohisto- Colorectal cancer'7 101 102 chemical methods, and with immunometric Crohn's disease'9 103 104 concentration measurements in various body Ulcerative colitis'9 105 106 Gastrointestinal mucosal inflammation'07 108 fluids (table 6). In several disorders the Urinary stone'09 differences between normal and pathological Oral inflammatory mucosal disease84 levels are large enough to suggest a diagnostic CNS inflammatory disease (multiple sclerosis and acute encephalitis) 10l potential for calprotectin. 15 17 18 97 105 106 118 Secondary CNS infections in HIV infected patients'8 Early attention was paid to rheumatic HIV infected patients7 l diseases.'3 .. .. Cystic fibrosis was studied Haematological patients50 Febrile conditions infectious and non-infectious18 before the protein was identified and character- Acute myocardial infarction"2 ised, and several other inflammatory diseases Surgery' 14-116 have since been studied (table 5). The Apheresis' 1' relevance of calprotectin in malignant diseases The references cited are representative reports or reviews, not a has also been pointed out.'6 17 50 123 complete list of publications on each topic. 116 6Johne, Fagerhol, Lyberg, Prydz, Brandtzeeg, Naess-Andresen, et al

Table 6 Calprotectin concentrations in various bodyfluids

Bodyfluid References Calprotectin concentrations* Serum/plasma Sander et al'5 Infectious diseases Normal 0.1-0.6 Mol Path: first published as 10.1136/mp.50.3.113 on 1 June 1997. Downloaded from Viral 0.1-1.4 Bacterial 0.6-11.0 Muller et al"9 HIV infection (25-75 percentiles) 1.2-9.4 Dale"3 Reference intervals Females 0.09-0.53 Males 0.12-0.66 Berntzen et al'2"14 Rheumatic disease Normal range 0.8-0.91 Rheumatoid arthritis 1-46 Osteoarthritis 0.5-0.8 Juvenile rheumatoid arthritis 2-24 Haga et al6 Systemic lupus erythematosus, mean 3.6 Semb et al"4 Cardiopulmonary bypass operations Preoperative 0.3 Postoperative 5.2±1.3 Garred et al"' Major surgery Preoperative baseline 0.5-0.9 Postoperative (4h) 7-15 Lugering 103 106 Crohn's disease Active 17±6 (MRP 8/14) Inactive 5±2 (MRP 8/14) Golden et al' Cystic fibrosis children Controls 0.3-1.6 (median 0.7) Cystic fibrosis 0.4-26 (median 1.8) Ivanov et alP Haematology Healthy blood donors 0.2 Leukaemia 0.4-13.3 (MRP 8/14) Cerebrospinal fluid Dunlop et al" Normal range 0-37 ,sg/l HIV positive with infections 30-350,g/l Oral fluids Brun et al' Sj0gren's syndrome Stimulated whole saliva 23.6 Cuida et al'2' Healthy subjects Parotis saliva 3.2 Stimulated whole saliva 22.0 Mucosal transudate 40.9 Muller et al"' HIV infected with or without oral candidiasis Parotis 0.06-0.41 Urine Holt et al'20 Infants and children (range) Controls 24 pg/l (5-650) Cystitis 182 sg/l (18-992) Pyelonephritis 1000 sg/ml (360-7000) Faeces Roseth et al'9 Median (range)

Healthy subjects 2 (0.5-8) http://mp.bmj.com/ Controls 10.5 (1.1-80) Crohn's disease 43 (8-2000) Ulcerative colitis 19.5 (2.4-866.4) Roseth et al" Colorectal cancer 50.0 (4.5-950) Gilbert et al'02 Healthy controls, range 0-9 Colorectal cancers (mean (SD)) Right side 55.1 (58.9) Left side 79.3 (58.2) Meling et al'09 Mean (95% confidence intervals) Normal baseline 4.9 (1.5-15.6) on September 29, 2021 by guest. Protected copyright. NSAID treated 9.0 (6-27) Synovial fluid Berntzen et al'4 Median (range) Rheumatoid arthritis 18 (2-375) Osteoarthritis 0.9 (0.2-2) Empyema fluid (supernatant) Santanagopalan et al'9 Not quantitated. Calprotectin functional assay 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 from have returned to baseline."16 128 129 Coronary citrated blood stored for 14-21 days. sequestration of both granulocytes and calpro- Increased plasma calprotectin concentra- tectin was demonstrated in the early reper- tions are found regularly in HIV infected indi- fusion period after cardiac surgery and cold viduals, and a strong further increase in cardioplegic arrest."4 response to zidovudine treatment seems to Recently, Arvesen et al"2 found that plasma predict a favourable prognosis. "' Curiously, calprotectin concentrations are increased in increased plasma calprotectin is associated patients with coronary artery disease. Even with a poor chance of survival in patients with patients with unstable angina or small (non-Q) alcoholic liver disease irrespective ofthe degree myocardial infarctions had mean values about of parenchymal liver damage.'27 three times the upper reference limit. An inter- During surgery, complement activation can esting possibility is that the elevations may be demonstrated by increased concentrations reflect an increased (inherited or acquired?) ofthe terminal complement complex or C3b in myelomonocytic response to even minor plasma. Simultaneously, granulocyte lysosomal stimuli that in the long run may predispose to proteins (myeloperoxidase and lactoferrin) and atherosclerosis. calprotectin increase, but the latter remains Plasma calprotectin levels are increased in elevated many hours longer than the former, active rheumatic diseases; Berntzen et al'2-14 even after the complement activation signals found 1-46 mg/l in rheumatoid arthritis Functional and clinical aspects of the myelomonocyte protein calprotectin 117

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 Mol Path: first published as 10.1136/mp.50.3.113 on 1 June 1997. Downloaded from Fulminant sepsis* 13.8 10.5-119.9 85 19-225 7.1 2.2-33.3 2800 210-170000 550 55-2400 Meningitis* 6.8 2.9-51.0 137 24-328 22.6 3.8-30.9 <3 <3-210 0 0-297 Mildsystmenin* 6.7 1.6-14.3 75 16-162 14.6 9.5-35.0 45 <3-600 3 0-102 Reference 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 meningococcal (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 protein (CRP). See also Brandtzeeg and Kierulf."3 patients and 2-24 mg/l in juvenile rheumatoid measurement has been suggested as a urinary arthritis, while Haga et ar6 reported a mean marker of asthma.'30 value of 3.6 mg/l in systemic lupus erythematosus patients compared with 1.05 mg/l in matched controls. In both rheumatoid arthritis CALPROTECTIN AND GASTROINTESTINAL DISEASES and systemic lupus erythematosus, plasma cal- R0seth et al'9 developed a method for the protectin values appear to be an objective determination of calprotectin in stool as an parameter for the assessment of disease activity alternative to al antitrypsin for the evaluation and response to treatment. Furthermore, both of disease activity in inflammatory bowel plasma and synovial fluid calprotectin clearly disease (IBD). It was, however, soon realised distinguish rheumatoid from osteoarthritis.'4 that increased faecal calprotectin is a marker of Plasma calprotectin concentrations are in- diseases of the gastrointestinal tract, including creased in endogenous posterior uveitis.95 gastric cancer, colorectal adenoma or cancer, Patients may also have raised antineutrophilic Crohn's disease, and ulcerative colitis. 10'9 105 cytoplasmatic antibodies (ANCA), but the The method depends on the preparation of a ANCA titres did not correlate with calprotectin simple buffer extract of a small spot sample of levels. The authors suggested the latter may be stool, and quantitation of calprotectin by a sensitive indicator of disease activity for enzyme linked immunosorbent assay (ELISA). endogenous posterior uveitis. There seems to Only a fraction ofthe total calprotectin content be a similar situation for cystic fibrosis.97 in the stool sample is brought into solution, and In a series of patients with newly diagnosed most of it is found in high molecular size com- pulmonary cancers,'23 increased plasma calpro- plexes (Roseth and Fagerhol, 1992, unpub- tectin was found in 81 %, while other disease lished).

indicating parameters lagged behind: CRP, In recently diagnosed colorectal cancers, http://mp.bmj.com/ 45%; orosomucoid, 48%; haptoglobulin, 59%; stool calprotectin concentrations above an a, antitrypsin, 31 %; alanine aminotransferase, upper reference limit of 10 mg/l were found in 5%; aspartate aminotransferase, 0%; y more than 90% of the patients.'9 In this study glutamyl transferase, 27%; alkaline phos- the value decreased below 10 mg/l after phatase, 10%. Strikingly, about 15% of the successful radical resection. No significant dif- patients had calprotectin concentrations (and ferences in calprotectin were found in cancers

other markers) close to or below the upper ref- of Duke's stages A and B compared with C and on September 29, 2021 by guest. Protected copyright. erence limit despite advanced, invasive disease. D, which suggests that a positive calprotectin This raises the possibility that some tumours test may be found even in early cancers. This is may release substances capable of inhibiting supported by the finding of increased concen- the emigration or activation ofmyelomonocytic trations in more than 60% of patients with cells. At least such cells would be expected to colorectal adenoma irrespective of size and participate in the removal of dead tumour cells. location.'0' Again, calprotectin concentrations returned to normal after removal of the CALPROTECTIN IN CEREBROSPINAL FLUID adenomas.'0' Although calprotectin is also syn- Limited data have been published, but calpro- thesised by squamous epithelial cells, it is not tectin levels in CSF seem to distinguish found in epithelial cells of the bowel nor in between HIV encephalitis and opportunistic colorectal cancer cells. The numbers of infections in the central nervous system in HIV infected neutrophils, however, are increased signifi- patients.'8 cantly in the peritumoral stroma in colonic CALPROTECTIN IN ORAL FLUIDS carcinomas.'3' The working hypothesis is that Concentrations between 3 and 40 mg/l have different types of pathological changes in the been measured in different oral secretions gastrointestinal tract cause increased perme- (table 6). Muller et al'l found lower calprotec- ability of the mucosa leading to increased tin concentrations in HIV infected patients migration of granulocytes and monocytes who suffered from oral candidiasis compared towards chemotactic substances in the gut with all HIV infected subjects, and with healthy lumen. An important factor for the high sensi- controls. tivity of the faecal calprotectin test is the fact that this protein is remarkably resistant to pro- CALPROTECTIN IN URINE teolysis in the presence of calcium.2 This may Increased calprotectin is found in patients with explain why the test has been found positive in urinary tract infections, particularly in patients more than 90% of patients with gastric with renal involvement.'20 Calprotectin cancer. 19 118 8Johne, Fagerhol, Lyberg, Prydz, Brandtzag, Naess-Andresen, et al

It is notable that faecal calprotectin shedding nohistochemical staining for calprotectin may may be caused by drugs'08 or other inflamma- be a diagnostic tool in inflammatory skin tory reactions in the gastrointestinal system. diseases, malignant conditions, and a variety of Thus, extensive clinical studies are being other diseases.3 133 Several commercial and conducted in this area, particularly aimed at non-commercial monoclonal antibodies are Mol Path: first published as 10.1136/mp.50.3.113 on 1 June 1997. Downloaded from confirming a negative predictive value for available (table 2). colorectal cancer. 7 Haemoglobin or faecal Monitoring of disease activity and therapy occult blood are used extensively as markers in may be relevant in inflammatory diseases such screening for colorectal cancer. Calprotectin is as rheumatoid arthritis, cystic fibrosis, Crohn's a less variable parameter than haemoglobin in disease, asthma,94 97 130 and others (tables 5 and faecal samples from patients with colorectal 6). cancers,'02 and although both parameters are Therapeutic use of calprotectin (or some of elevated significantly, they do not correlate. its structural elements) is a fascinating thought Thus the mechanism of luminal calprotectin with its protective biological properties. entry appears to be both different from and less Lehrer32 calls it "an internal ointment that erratic than bleeding.'02 restricts the growth of C albicans on nearby In patients with active IBD, faecal calprotec- skin." Could it be used as an external tin concentrations above 100 mg/l are regularly ointment? In terms of therapy could the found,'0' and a diagnosis of IBD should not be molecule be used in rheumatic, inflammatory, made in a symptomatic patient with a faecal infectious or malignant diseases? calprotectin level below 10 mg/l. Highly significant correlations were found between this test Unpublished data and the IBD disease activity determined by SYNTHESIS AND RELEASE OF CALPROTECTIN FROM endoscopy, histology or excretion of "'indium HUMAN MONOCYTES IN VITRO labelled autologous granulocytes."' 105 The lat- A series of experiments were performed imme- ter method has been regarded as the "gold diately after the discovery of calprotectin, to standard" but its use is limited by complexity, gather data concerning the mechanisms in- cost, and exposure of the patient to irradiation. volved in the release of calprotectin from The relatively simple faecal calprotectin test is monocytes. These data have not previously an attractive alternative as it can be repeated at been published, nor have similar data been any time and, because ofthe stability of calpro- published by others. We have therefore in- tectin, samples can be mailed by the patient to cluded them in the present review. the laboratory. Treatment response in patients with Crohn's disease can be monitored by fae- Materials and methods cal calprotectin measurements. Human monocytes were isolated from healthy fasting donors as described previously'34 35 and CONCLUSION cultured in RPMI 1640 (Gibco-Biocult, Pais- http://mp.bmj.com/ The clinical relevance of calprotectin seems to ley, Scotland) containing 20% inactivated fetal be related to its physiological functions during calf serum (Flow, Irvine, Scotland). The final homeostasis as well as in pathogenesis. These cell preparations consisted of more than 95% functions are intracellular as well as extracellu- monocytes demonstrated by differential count- lar. Disease modulation during rheumatoid ing, phagocytosis and staining for non-specific arthritis and possible antitumour effects of cal- esterase.'36 The presence of calprotectin was protectin may result from specific mechanisms determined by single radial diffusion or ELISA on September 29, 2021 by guest. Protected copyright. other than antimicrobial activity. Calprotectin after incubation periods of up to 46 hours. The is a non-specific marker for activation of synthesis of calprotectin was studied by addi- granulocytes and mononuclear phagocytes. It tion of 35S-methionine to the medium, and is released from these cells and is thus expected radiolabelled calprotectin was determined by to be found in secretory and excretory an immunoaffinity column with immobilised, products in different parts of the body. monospecific rabbit anti-calprotectin antibodies. Possible clinical applications The following compounds were added to The diagnostic value of calprotectin has been monocyte cultures: immune complexes formed emphasised in several publications (tables 5 by incubating equivalent amounts of human and 6) that recommend calprotectin as an serum albumin or transferrin (Sigma, St Louis, inflammatory disease marker. Extensive and Missouri, USA) and corresponding antibodies systematic clinical documentation is still lack- (Dakopatts, Copenhagen, Denmark); aggre- ing, however, some larger studies are ongoing. gated human IgG (Gammaglobulin, Kabi, RIA"3 and ELISA' have been established as Stockholm, Sweden) prepared by heating a 1% immunoassays for calprotectin measurement in solution in 0.15 M NaCl at 63°C for 12 body fluids, and are also applied to stool minutes, and precipitation with Na2SO4 (67 g/l); samples."'' Two commercial ELISA assays cross-linked human immunoglobulins prepared exist, with an analytical sensitivity at the ng/ml by reacting IgG (10 g/l) with 0.5% glutaralde- level. In the NycoCard (Nycomed, Oslo, Nor- hyde in 0.1 M acetate buffer, pH 4.0 for one way) immunoassay format'25 132 a prototype hour at room temperature, followed by dialysis quantitative rapid test for calprotectin has been against distilled water and phosphate buffered established with an analytical sensitivity of saline, pH 7.35; concanavalin A (ConA) and approximately 0.05 gg/ml. Documentation will wheat germ agglutinin (WGA) (Sigma); phyto- increase as commercial calprotectin tests be- haemaglutinin (PHA) (Wellcome, Beckenham, come easier and more widely available. Immu- Kent, UK); ionophores A 23187 and nigericin Functional and clinical aspects of the myelomonocyte protein calprotectin 119

Table 8 Release ofcalprotectin from human monocytes after 16 hours in vitro culture (Lilly Research Corporation, Indianapolis, Indi- ana, USA); endotoxins E coli 0 11 :B4 and E coli Concentration Serum * No. of Calprotectin Stimulant ,uglml 20% cultures releaset (ng 1ml) 055:B5 (Difco, Detroit, Michigan, USA); ac- - tinomycin D (Serva, Heidelberg, Germany); Lectins Mol Path: first published as 10.1136/mp.50.3.113 on 1 June 1997. Downloaded from ConA 25 + 14 < 25 1 2-O-tetradecanoylphorbol- 13-acetate (TPA) ConA 25 - 2 98 (PL Biochemicals, Milwakee, Wisconsin, USA); PHA 25 + 8 49 cycloheximide, cytochalasin B, and prostaglan- PHA 25 - 2 130 WGA 25 + 4 46 din El (PGE,) (Sigma). WGA 25 - 2 1050 Lactate dehydrogenase, lysozyme, and P glu- Antigen/antibody complexes coronidase activities were determined in cul- HSA/anti-HSA 10t + 8 665 HSA/anti-HSA 10t - 2 265 ture supernatants and cell homogenates.'37-139 Transferrin/antitransferrin 2t + 6 440 Heat aggregated IgG (IgG-H) 700 + 2 15 Results Glutaraldehyde aggregated IgG (IgG-G) 700 + 2 25 Freshly prepared monocytes contained HSA/anti-HSA + IgG-H 10t + 700 + 2 48 2.5 ± 0.7 pg calprotectin per cell, and after HSA/anti-HSA + IgG-H 10* + 250 + 2 52 incubation for 9-10 hours the content was HSA/anti-HSA + IgG-G 10t + 700 + 2 37 HSA/anti-HSA + IgG-G 10* + 250 + 2 49 2.3 ± 0.2 pg/cell. Ionophores Various agents known to interact with and A 23187 0,25 + 14 165 monocytes were added to monocyte A 23187 0,25 - 6 319 activate'40 Nigericin 2,5 + 2 > 1000 cultures. None of these, except TPA at 1 ptg/ml Endotoxin 25 + 12 136 caused cytotoxicity (increased trypan blue Phorbol ester uptake) within the time period studied. As TPA 1 + 2 2238 shown in table 8, very small amounts of calpro- * Fetal calf serum inactivated at 56'C for 30 min. tectin were released from monocytes cultured t The release from unstimulated control cells was < 25 nglml. for 16 hours in the presence of lectins and 20% * Amount of antigen present in the immune complex added. HSA, human serum albumin. inactivated fetal calf serum. In the absence of serum slightly higher amounts (but less than Table 9 Release oflysozyme and /3 glucuronidase from stimulated human monocytesi;*n 1% of total cellular calprotectin content) were vitro released after ConA or PHA stimulation, while WGA caused a noticeably higher release. No Lysozyme % of fi glucuronidase % of release of lactate dehydrogenase or : glucuro- unstimulated unstimulated control Stimulant control (SEM) Cultures (n) (SEM) Cultures (ien) nidase, and no increased lysozyme release was seen in lectin stimulated cultures (table 9). Immune complexes 695 (189) 12 610 (79) 8 Endotoxin 138 (45) 10 107 (4) 8 Immune complexes led to a marked release of A 23187 435 (251) 12 198 (68) 10 calprotectin over two to three hours (fig 1), and Lectins 92 (8) 16 97 (21) 14 about 8% of the calculated total cellular

Nigericin 257 2 356 2 http://mp.bmj.com/ TPA 664 (114) 10 425 (70) 10 calprotectin was released. This release was two to threefold higher in the presence of fetal calf * The final concentrations of stimulants were as given in table 8. serum (table 8). Native serum was 25% more active than inactivated serum, and native serum alone promoted the release of calprotec- 1000 tin in the absence of immune complexes. Addi- tion of immune complexes induced a marked release of both ,B glucuronidase and lysozyme on September 29, 2021 by guest. Protected copyright. (table 9), but essentially no release of lactate dehydrogenase. Phorbol ester (TPA) at 500 ng/ml caused a quite rapid and marked release of calprotectin (about 7% of total cellular content), but no 100 signs of cytotoxicity in three hours (fig 1). Release of the total intracellular calprotectin C was seen when a cytotoxic dose of TPA (1 gg/ c)0 ml) was applied for 14 hours (table 8). The 0. divalent ionophore A23 187 caused a moderate (o release of calprotectin, and the monovalent ionophore nigericin a substantial release (table 20 8). Both ionophores increased moderately the 10 O TPA, 0.5 pg/ml release of ,B glucuronidase and lysozyme (table a IC, 10 pg/mI 9). A LPS, 25 pg/mI Endotoxin induced a moderate but distinct release of calprotectin (table 8), but no significant increase in lysozyme or f glucuronidase secretion (table 9). A very small amount (less than 2% of total cellular content) was released 1 during the first three hours (fig 1). 0 50 100 150 200 To learn more about the mechanism of Minutes calprotectin release, addition of several other were tested. E, Figure 1 Time course for release ofcalprotectin from human monocytes in vitro. compounds Prostaglandin TPA, 12-0-tetradecanoylphorbol-13-acetate; LPS, endotoxin; IC, immune complexes. The (10 jM) did not influence the release of final concentrations in pglml of the added substances are indicated. calprotectin during incubation with TPA. The 12010ohne, Fagerhol, Lyberg, Prydz, Brandtzag, Naess-Andresen, et al

Table 10 Release ofcalprotectin, lysozyme, andfi glucuronidase from stimulated human although not exactly in the same proportion. A monocytes in vitro, with potentially interfering substances added correlation was observed between lysozyme and calprotectin release. The effects of dexa- Stimulant* Addinonst Calprotectint Lysozyme fi glucuronidaset methasone served to distinguish these pro- Immune complexes Dexamethasone 101 (7) 84 (7) 87 (35) cesses in that it reduced calprotectin release Mol Path: first published as 10.1136/mp.50.3.113 on 1 June 1997. Downloaded from Promethazine 98 131 134 Indomethacin 81 (12) 100 (5) 115 (8) while leaving lysozyme and 1 glucuronidase Endotoxin Dexamethasone 44 (6) 99 (15) 141 release unchanged in response to endotoxin. In Promethazine 248 (70) 1250 597 (260) the presence of lectins or A23187 the opposite A 23187 Dexamethasone 122 93 (7) 18 Promethazine 311 452 426 was found: dexamethasone reduced 13 glucuro- Indomethacin 196 (12) nidase release whereas calprotectin and lys- Lectins Dexamethasone 95 (25) 101 (18) 58 (15) ozyme release were unchanged. It is therefore Indomethacin 230 likely that calprotectin release is different from * The final concentrations of stimulants were as given in table 8. the lysozyme and lysosomal release processes. t Final concentrations: dexamethasone, 10 g/ml; promethazine and indomethacin; 0.1 mM. Clearly, calprotectin release can be caused by a t Each value is the mean of 2-8 cultures, and calculated as % of stimulated control (SEM). number of membrane perturbing agents, and prostaglandin synthesis inhibitors indometh- calprotectin is either derived from the plasma acin and acetylsalicylic acid had no effect on membrane or from an intracellular compart- the release caused by immune complexes or ment by some special release process.'42 lectins (table 10). Release induced by A23187 Anti-calprotectin antibodies added to was enchanced twofold by indomethacin but human monocytes in culture induced the syn- not by acetylsalicylic acid. As indomethacin has thesis of the protein component of other effects besides blocking prostaglandin thromboplastin,'40 which suggests that calpro- synthesis,14' our tentative conclusion is that tectin is at least in part located in the plasma products of the prostaglandin pathway are not membrane, as observed by Dale et ar' and involved in calprotectin secretion. The antihis- Guignard et al.88 tamine promethazine enhanced the release of calprotectin induced by endotoxin, A23187 or Conclusions TPA (data not shown), whereas the release Calprotectin is fascinating because it holds induced by immune complexes was unchanged unrevealed secrets. We know that calprotectin (table 10). Similarly, both lysozyme and 1 glu- is an important granulocyte marker and a multi- curonidase release was enchanced by addition functional regulatory protein in inflammatory of promethazine to A23187 or endotoxins, but processes. There is more work to do in order to not to immune complex stimulated monocytes understand fully basic mechanisms such as (table 9). Dexamethasone inhibited calprotec- quaternary structure, complex assembly, mo- tin release induced by endotoxins but not that lecular functions, and biological effect mecha- induced by A23187, lectins or immune com- nisms. plexes. This drug had no effect on the lysozyme Much has been reported regarding the clini- http://mp.bmj.com/ release, but the release of 1 glucuronidase cal relevance and diagnostic potential of induced by A23187 and lectins was inhibited. calprotectin and its subunits, and larger clinical Cytochalasin B (10 jtg/ml), which interferes studies are continuing. Further work may with actin polymerisation, had no effect on cal- gather speed as commercial kits for calprotec- protectin release induced by endotoxin, but a tin measurement become more readily avail- twofold enhancing effect in the presence of able. Interesting therapeutic concepts may be PHA. found for calprotectin, as the relation of the on September 29, 2021 by guest. Protected copyright. Dexamethasone (10 jg/ml), dibutyryl cAMP structure and function of the molecule are bet- (1 mM), cAMP (1 mM), verapamil (25 jg/ ter understood. ml), acetylsalicylic acid (0.1 mM), and purified In the meantime we will do as Lehrer" protein derivative of Bacillus Calmette-Guerin recommended in 1993: "Stay tuned. Calpro- (PPD) had no effect of calprotectin release tectin, whatever it is, could be interesting." from unstimulated cells. 1 Fagerhol MK, Anderson KB, Naess-Andresen CF, Brandtzxg P, Dale I. Calprotectin (the LI leukocyte Discussion orotein). In: Smith VL, Dedman JR, eds. Stimulus response coupling: the role ofintracellular calcium-bindingproteins. Boca The release of calprotectin from isolated Raton: CRC Press Inc, 1990:187-210. monocytes can result from general cytotoxicity. 2 Fagerhol MK. Nomenclature for proteins: is calprotectin a proper name for the elusive myelomonocytic protein? Jf Clin If sufficiently high doses are used-for exam- Pathol: Mol Pathol 1996;49:M74-9. ple, TPA at 1 ,ug/ml, the total calprotectin pool 3 Brandtzwg P, Dale I, Gabrielsen TO. The leukocyte protein Li (calprotectin): usefulness as an immunohistochemical may be released. However, partial release was marker antigen and putative biological function. Histopa- observed without concomitant lactate dehy- thology 1992;21:191-6. 4 Brandtzmg P, Gabrielsen T-0, Dale I, Muller F, Steinbakk drogenase release or trypan blue uptake. This M, Fagerhol MK. The leukocyte protein LI (calprotectin): suggests that either calprotectin release is a a putative nonspecific defence factor at epithelial surfaces. Adv Exp Med Biol 1995;371:201-6. very sensitive indicator of cytotoxicity or the 5 Sorg C. The calcium binding proteins MRP8 and MRP14 release is caused by other mechanisms. in acute and chronic inflammation. Behringer Inst Mitt 1992;91:126-37. There was no definite evidence for involve- 6 Brun JG, Ulvestad E, Fagerhol MK, Jonsson R. Effects of ment of the prostaglandin system in calprotec- human calprotectin (LI) on in vitro immunoglobulin synthesis. Scand J Immunol 1994;40:675-80. tin release, and the fraction released during 7 McNamara MP, Weissner JH, Collins-Lech C, Hahn JH, three to four hours never exceeded 8% of the Sohnle PG. Neutrophil death as a defence mechanism against Candida albicans infections. Lancet 1988;ii: 1163- total cellular pool. 5. The agents that promoted lysosomal enzyme 8 Steinbakk M, Naess-Andresen C-F, Lingaas E, Dale I, Brandtzaeg P, Fagerhol MK. Antimicrobial actions of release most (immune complexes, TPA, iono- calcium binding leucocyte Li protein, calprotectin. Lancet phores) also increased the calprotectin release, 1990;336:763-5. Functional and clinical aspects of the myelomonocyte protein calprotectin 121

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