JOURNAL OF PATHOLOGY, VOL. 180: 194-199 (1996)

EXPRESSION OF THE Ll ANTIGEN (CALPROTECTIN) BY TISSUE MACROPHAGES REFLECTS RECENT

RECRUITMENT FROM PERIPHERAL BLOOD RATHER THAN UPREGULATION OF LOCAL SYNTHESIS: IMPLICATIONS FOR REJECTION DIAGNOSIS IN

FORMALIN-FIXED KIDNEY SPECIMENS

JARLE RUGTVEIT*, HELGE SCOTT*, TROND S. HALSTENSEN*?, JARLE NORSTEIN: AND PER BRANDTZAEG*

* LIIPA T. Itistitute of’ Puthology and ZSzrrgical Depurttnent B, University qf Oslo, The Nutionul Hospitul, Rikshospitulet, N-00;?7 Oslo, Norwuy; +MIMI, Nutionul Institute of Public Health, N-0462 Oslo, Norwuy

SUMMARY

The L1 antigen (calprotectin) is present in circulating monocytes but is restricted to certain subsets of tissue macrophages. Its expression is significantly increased in inflammatory bowel disease, apparently because of newly recruited monocytes. I n v i m experiments were performed to substantiate lack of L l upregulation in tissue macrophages, thereby justifying the use of this marker to detect newly recruited cells. Its reliability was further evaluated by studying mononuclear cell infiltrates characteristic of acute kidney rejection. After pro-inflammatory stimulation, monocytes matured in V ~ ~ J W ( I # = 12) as well as adherent mononuclear cells from normal small intestinal mucosa (n=5) were examined for L l expression by immunocytochemistry and by ELISA (cell lysates). In addition, peritubular mononuclear L 1 + cells were examined by immunohistochemistry in routine biopsy specimens from transplanted kidneys with (n= 11) or without (n= 14) histopathologically diagnosed acute rejection. L1 was not upregulated in monocytes matured in vifro, nor in mucosal macrophages after stimulation with interferon-y, LPS, phorbol ester, or supernatant from activated leucocytes. In transplanted kidneys with signs of acute rejection, the fraction of Ll + macrophages was significantly increased (P<O*OOI). Because Ll is persistently downregulated in mature tissue macrophages and is formalin-resistant, it identifies young infiltrating macrophages in routinely processed biopsy material. Ll should therefore be a valuable adjunct in the diagnosis of kidney rejection.

KEY WORDS-Ll antigen; calprotectin; MRP-8/12; macrophage; monocyte; kidney transplantation; rejection

INTRODUCTION

The L1 antigen (calprotectin) is a major cytosol protein complex present in virtually all circulating monocytcs and neutrophil granulocytes. It is restricted to certain subsets of tissue macrophages in inflammatory lesions.’ LI consists of two subunits (Ll, and LI, chains) which are identical to the proteins called MRP-8 and MRP-14.? Its alternative name, calprotectin, refers to the antimicrobial activity shown by the L1 ~ o m p l e x . ~ Antiproliferative properties against tuinour cell lines have been reported as we1L8 I t might also be of importance for the activation, motility, and recruitment of nionocytes.”-“) Altogether, the abundant expression of this protein by inflammatory leucocytes, including reactive macrophages, may bc of biological significance in local defence.

We recently showed that mucosal macrophages in inflammatory bowel disease (IBD) contain an increased L1+ fraction; these cells were probably newly recruited from peripheral blood monocytes. Moreover, the LI, and Ll,, subunits have been reported to be down- regulated in nioiiocytes that mature in suggesting expression only at an early stage of macrophage

Addiesscc for correspondence J Rugtveit, LIIPAT, Rik\ho?pttalet, N-0027 Odo. Noiw‘iy

CCC 0022-34 1 71961 100 1 94-06 c 1996 by John Wiley & Sons. Ltd

differentiation. Because L l is a formalin-resistant antigeq2 it might be considered as a useful marker for young macrophages in routinely processed biopsy material. A requirement would be that it is not upregu- lated in resident macrophages in inflamed tissue. Such upregulation of L1 takes place in keratinocytes in inflammatory dermatoses,I4 suggesting that de n o w syn- thesis of LI is possible in differentiated cells exposed to cytokines or other regulatory factors in inflamed tissue.

In this study we examined whether the synthesis of L1 can be upregulated in isolated tissue macrophages or monocytes matured in v i m . Because mononuclear cells appear early in kidney graft rejection,I5.I6 macrophages belonging to peritubular infiltrates of the acute reaction must be recently extravasated or ‘young’ cells. We therefore studied L 1 expression in biopsy specimens from transplanted kidneys clinically suspected to have acute rejection reaction, compared with counterparts deemed to have older lesions. The combined infor- mation derived from these two test models should add to our knowledge of the value of LI as a dynamic macrophage marker.

MATERIALS AND METHODS Blood mononuclear celh

Monocytes from 12 healthy blood donors were isolated by separation on Lymphoprep (Nycomed, Oslo,

Recrivetl 15 November 1995 Acceptid 19 Fcdmuy I996

LI-EXPRESSING TISSUE MACROPHAGES 195

Norway) and subsequent adherence to tissue chamber slides (LAB-Tek, Nunc, Roskilde, Denmark) or 96-well microtitre tissue culture trays (Multiwell, Falcon, Becton Dickinson, Mountain View, CA, U.S.A.). The adherent cells were cultured in RPMI-1640 with 10 per cent human serum (Sigma, St. Louis, MO, U.S.A.) for 10 days, the last two days with phorbol myristate acetate (PMA, 200 mM) (n = 6), supernatant of autologous cul- tures of peripheral blood mononuclear cells (PBMNC) stimulated with phytohaemaglutinin (PHA, 10 ,ug/ml) for 24 h (n=6) or interferon-? (IFN-.J; Genzyme, Cambridge, MA, U.S.A.) at 200 U/ml (n= 12), the latter with (n=6) or without (n=6) simultaneous addition of lipopolysaccharide (LPS, from Escherichiu coli Serotype 026 B:6; Sigma) at 1 puglml. Penicillin (100 U/ml), streptomycin (100 mg/ml), gentamycin (40 mg/ml), and L-glutamine (2 mmol/l) were added to all media.

Intestinal mononuclear cells

Adherent lamina propria mononuclear cells (LPMNC) were isolated from normal small intestinal mucosa of five organ donors with artificially maintained peripheral circulation. A modification of the EDTA- collagenase technique of Bull and BookmanI7 was used for cell dispersion, followed by adherence to micro- titre wells or chamber slides coated with 0.1 per cent gelatin (Sigma) and 50 per cent inactivated human plasma.18 The adherent cells were cultured for 3 days with 1000 U/ml granulocyte-macrophage colony- stimulating factor (GM-CSF) (kindly provided by Shering-Plough Research, Bloomfield, NJ, U.S.A.), the last two days with addition of IFN-7 at 200 U/ml with (n=5) or without (n=4) LPS at 1 yglml.

Characteristics of cell cultures

The fraction of monocytes in 8-day-old PBMNC adherent cultures was median 93 per cent (range 90-95 per cent) as determined by butyrate esterase staining, and all adherent cells were considered viable. The viability of the LPMNC after density gradient centrifu- gation was median 92 per cent (range 90-96 per cent) determined by staining with acridine orange/ethidium bromide. l9 The fraction of macrophages after adherence separation was median 16 per cent (range 15-18 per cent). Adherent cells were again taken to be 100 per cent viable. At the start of stimulation, there was less than 0.1 per cent contamination of granulocytes in the adherence-separated LPMNC as judged by nuclear staining. The maturation of the monocytes was evalu- ated by means of monoclonal antibodies (MAbs) Max 1, Max 2, and Max 11 (kindly provided by Dr Andreesen, Regensburg, Germany) against maturation antigens2" in a cell-ELISA method described previously.21

Quantification of L l by ELISA

For quantification of L1 in cultured mononuclear cells, the microtitre trays were centrifuged, the super- natant was discarded, and 200,ul of PBS was added per well. The cells were then subjected to three freeze-thaw

cycles before L1 in the lysates of stimulated and unstimulated cells was quantified by ELISA as described elsewhere.22 Determination of the cell numbers in the microtitre trays was performed in identically treated parallel wells with a modification of the method described by Gillies et 01.~'

Kidney tissue specimens

Routine biopsy specimens fixed in buffered formalin (pH 7.4) were obtained from transplanted kidneys; they were judged independently by two experienced histo- pathologists to show signs of acute rejection (n= 11) according to the BANFF criteria,24 or to have older inflammatory cell infiltrates (n= 14). In addition, formalin-fixed and paraffin-embedded operation speci- mens from end-stage kidneys (n=9) were included. The fraction of L1+ mononuclear leucocytes and the pro- portion of L1+ macrophages present in the tubulo- interstitial mononuclear cell infiltrates were evaluated, respectively, by applying the alkaline phosphatase/ anti-alkaline phosphatase (APAAP) or the two-colour immunofluorescence staining method described below.

APAAP staining

Kidney tissue sections, cut at 5 pm, were dewaxed and treated with trypsin (1 g/l) for 10 min at 37°C and incubated for 12 h with the MAb against L1 (Mac 387; DAKO, Glostrup, Denmark) at 0.02,ug IgGlml or with MAb against CD45 or leucocyte common antigen (DAKO) at 8.1 ,ug IgG/ml, followed by rinsing in Tris- buffered isotonic saline (pH 7.6) for 4-5 min. The antibody binding sites were then detected by APAAP staining performed according to Mason.25 Isotype- and concentration-niatched MAb against irrelevant antigen (mouse IgG1 against keyhole limpet cyanin, Becton Dickinson) was used as a negative control. Mac 387 had to be carefully titrated to the working concentration indicated above to optimize distinction between macro- phages with high and low or no L1 expression; under these conditions, the APAAP results obtained harmo- nized with the identification of L1+ and LI - cells made by the direct immunofluorescence staining with a rabbit IgG-rhodamine conjugate (see below).

Two-colour immunofluorescence staining

Adherent PBMNC and LPMNC on chamber slides were fixed at 4°C for 10 min in 4 per cent (w/v) paraformaldehyde and incubated for 1 h with rabbit IgG (35 ,ug/ml)-tetramethylrhodamine (TRITC) conju- gate specific for the L1 antigen' mixed with MAb KP1 (supernatant, 1/50; courtesy of Dr D. Y. Mason, Oxford, U.K.) against the myelomonocytic marker CD68.26 This combination was followed by incubation with biotinylated horse anti-mouse IgG (25 p g IgG/ml, Vector Laboratories, CA, U.S.A.) for 1.5 h, and finally fluorescein isothiocyanate (F1TC)-labelled streptavidin (Boehringer Mannheim Biochemica, D-6800 Mannheim, Germany) at 20pglml for 30 min.

196 .J. RUGTVEIT ET AL.

Dewaxed and trypsinized tissue sections were incu- bated for 12 h with TRITC-conjugated anti-Ll mixed with MAb PG-MI (1Spg IgG/ml, DAKO). This MAb shows restricted specificity for CD68 and does not react with granulocytes. Biotinylated horse anti-mouse IgG antibody and FITC-labelled streptavidin were finally applied as above.

TRITC-labelled rabbit IgG from normal rabbit serum was applied as a negative control for the anti-Ll conju- gate at the same concentration and with the same degree of conjugation."

Evaluation of LI expression in kidney sections

The areas with the densest peritubular mononuclear cell infiltrates were evaluated in each section (see below). At least 200 CD68+ cells were examined for co-expression of L1 by two-colour immunofluorescence staining at x 400 magnification in a Leitz DMRDXE microscope equipped with a Ploem-type vertical illumi- nator and a Leitz DMRD camera system (Leitz, Wetzlar, Germany). Switching of the filter sets allowed selective observation of individual cells with regard to TRITC (red emission) or FITC (green emission). Double-exposed microphotographs were taken on Ektachrome 800/1600 I S 0 daylight film pushed to 800 I S 0 for fluorescence microscopy.

I n comparable areas of serial tissue sections, the overall cellular density of the peritubular infiltrates was recorded on an arbitrary scale as moderate or high by APAAP staining for CD45. In corresponding fields of the adjacent section, the fraction of L l + cells was estimated or scored semi-quantitatively as <25 per cent, 25-50 per cent or >50 per cent, compared with the observed density of CD45' cells. Microscopical evalu- ations were performed independently by two blinded observers for both staining methods; no significant inter- observer difference was found, either in the evaluation of the fraction of CD68' cells expressing L1 in the peri- tubular infiltrates by two-colour immunofluorescence staining (Wilcoxon signed-rank test) or in the semi- quantitative scoring of the fraction of L1+ mononuclear leucocytes by the APAAP method (same score in 22 of the 25 specimens from transplanted kidneys).

Statistics

Data were treated by non-parametric methods. The Wilcoxon signed rank test or Mann-Whitney U-test

(two-tailed) was used for paired or unpaired group comparisons, respectively.

RESULTS

Cell cultures Significantly increased expression of maturation anti-

gens identified by MAbs Max 1 (P<O.Ol), Max 3 (P=O.OI), or Max 11 (P=0.02) was revealed in in vitro-matured monocytes cultured for 7 days, compared with the freshly prepared counterparts. Furthermore, L i expression decreased from median 97 per cent (range 96-100 per cent) on the first day to median 2,5 per cent (range 0-9 per cent) after 10 days in culture. Adherent isolated intestinal macrophages from normal mucosa showed only median 5 per cent (range 2-9 per cent) L1 expression. No significant increase in the percentage of L1' cells occurred after stimulation of the adherent monocyte cultures for 2 days with PMA, with supernatants from autologous cultures of PHA- stimulated PBMNC, or with IFN-y in the presence or absence of LPS. Nor was there any increase in the percentage of Li' intestinal macrophages ( < 5 per cent) after 2 days of stimulation with TFN-7, with or without addition of LPS.

Tissue sections

Transplanted kidneys with signs of acute rejection contained a prominent L1' subset (median 59.5 per cent; range 20-85 per cent) among the CD68' macro- phages in the tubulo-interstitial infiltrates (Figs 1 a and ib). The L1+ fraction was significantly higher (P<O.OOI) than in comparable infiltrates in transplanted kidneys without evident rejection activity (Figs 2a and 3), where the value was 11.0 per cent (range 3-27 per cent), as well as in end-stage kidneys (P<O.OOl ) with 10.5 per cent (range 3-18 per cent). Only a few scattered L1+ cells appeared to be granulocytes in kidneys with acute rejection, as they were not stained by the macrophage- specific MAb PG-M1 against CD68.

Furthermore, tubulo-interstitial mononuclear cell infiltrates evaluated by APAAP staining for L1 and CD45 in adjacent tissue sections from kidney specimens with acute rejection generally contained a much higher fraction of L1+ cells (Figs l c and Id) than comparable infiltrates in tubular areas of transplanted kidneys without histopathological signs of rejection (Figs 2b

Fig. I-Mononuclcar cell inliltrate in comparable peritubular areas from trypsinized serial sections of a formahf ixed biopsy specimen from a kidney with histopathologically diagnosed acute rejection. (a) Two-colour immunofluorcscence staining (double exposure) for CD68 (FITC, green) and L1 antigen (TRITC, red) shows that only a few scattered cells lack LI (pure green colour) (original magnification x 400). (b) The same staining shows mononuclear cells surrounding a tubular structure; in this field, about half of the cells are double-positive (grades of yellow) (original magnification x 1000). ( c ) Comparable peritubular area as in (a) from a serial section stained by APAAP for CD45 (original magnification x 400). (d) Comparable field in an adjacent scction stained for LI shows massive positivity in the peritubular infiltrate and some in the glomerulus (original niagnilication x 400)

Fig. 2-Mononuclear cell infiltrate in peritubular areas in trypsinized serial sections of a formalin-fixed biopsy specimen from a transplanted kidney with no histopathological signs of acute rejection. (a) Two-colour immunofluorescence staining (double exposure) for CD68 (FITC, green) and L I antigen (TRITC. red) shows only a few cells with co-expression of the two markers (yellow). (b) (:omparable area in a serial section stained by APAAP for CD45' shows numerous mononuclear cells. (c) Comparable field in an adjacent section stained by APAAP for LI shows only a few positive cells (original magnification x 400)

198 J. RUGTVEIT ET AL.

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Fig. 3-Percentage of LI + macrophages in peritubular mononuclear cell infiltrates of transplanted kidneys with histopathological signs of acute rejection ( 0 ) or no active reaction (0). Areas with the highest mononuclear cell density within each section were evaluated by paired immunofluorescence staining for Ll and CD68 (see Figs l a and 2a)

and 2c); the LI + fraction in the latter apparently older infiltrates was semi-quantitatively estimated (see Materials and Methods section) to be below 25 per cent in 12 of the 14 specimens, whereas in all specimens with acute rejection the fraction was above that level (data not shown).

DISCUSSION

Monocytes matured in vitro showed downregulation of L1 as previously reported for its two subunits.” Various macrophage activators, including IFN-y and LPS combined, neither retrieved LI expression in such cells, nor increased it in isolated macrophages from normal intestinal mucosa. We have obtained upregu- lation of IL-1p after similar stimulation of intestinal macrophages. from patients with IBD, suggesting that the isolation and culture conditions used in this study did not cause a general cellular unresponsiveness (Rugtveit et a]., in preparation). Although lack of L1 expression by mature macrophages in vitro does not exclude the possibility that an upregulation of this marker may occur in vivo, our findings support the view that L1+ mononuclear cells in inflammatory lesions are young rather than resident macrophages.’ This conclusion was strengthened by our finding of a large fraction of L1+ macrophages in tubulo-interstitial mononuclear cell infiltrates in the transplanted kidneys with acute rejection reactions, in striking contrast to that observed in older lesions found in transplanted kidneys without histopathological signs of rejection.

In active mucosal lesions of patients with IBD, we have previously found that LI is absent in the predomi- nant macrophage population decorated by RFD7.”,** This MAb identifies resident macrophages and reacts with in vitro-matured m o n ~ c y t e s . ~ ~ High expression of

both LI subunits was furthermore observed in mouse peritoneal macrophages a few hours after the induction of an acute inflammatory reaction, in contrast to the lack of such expression 4 days later.’O Although there might be mitotic activity in some of the infiltrating macrophages recently derived from peripheral blood, local proliferation is unlikely to contribute a substantial fraction of the immature L1+ subset seen in active inflammatory lesions.31

The usefulness of L1 as a macrophage marker depends on the actual tissue preparation. Extensive leaching of this protein from sections not subjected to cross-linking fixatives makes evaluation of cellular L1 expression, including possible differential regulation of the two and local LI secretion, quite unre- liable.2T33 It is important for diagnostic purposes that L1 is remarkably well preserved within individual cells in formalin-fixed and paraffin-embedded routine biopsy material. Macrophages that express LI can be differen- tiated from L1’- granulocytes by paired immunohisto- chemical staining for the monocyte/macrophage-specific formalin-resistant CD68 marker detected by the commercial MA b PG-M 1 .34

In addition to lymphocytes, macrophages appear to be a major part of the mononuclear cell infiltrate that characterizes acute kidney rejection.I5.l6 This fact, how- ever, has until recently received only limited attention. The literature focusing on monocyte/macrophage phe- notypes in graft rejection is relatively sparse, probably due in part to the lack of suitable markers.’s A few reports have emerged applying antisera or MAbs reactive against infiltrating macrophages or their subsets,36-39 but none of these studies was performed on routine biopsy material. To our knowledge, LI is the only dynamic formalin-resistant myelomonocytic marker, being downregulated in mature tissue macro- phages, except for some epithelioid and giant cells.* It is therefore an ideal marker for recently extravasated or young macrophages in formalin-fixed, paraffin- embedded routine material. In our opinion, LI expres- sion should aid the estimation of the age of mononuclear cell infiltrates; this study suggests that it could be a valuable adjunct in the diagnosis of kidney rejection, by distinguishing acute reactions from older cellular infiltrates.

ACKNOWLEDGEMENTS

We acknowledge Dr Inge Dale for assistance in the quantification of L1 (calprotectin) by ELISA. The tech- nical staff of LIIPAT are thanked for excellent cooper- ation. This work was supported by grants from the Research Council of Norway, the Norwegian Cancer Society, Anders Jahre’s Fund, Medinnova Governmen- tal Research Organization, and Familien Blix’ Fond. J.R. is a Research Fellow of the University of Oslo.

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LI-EXPRESSING TISSUE MACROPHAGES 199

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