Tissue distribution of the human CD97 EGF ... - Wiley Online Library

L.H. Jaspars W. Vos G. Aust R.A.W. van Lier J. Hamann

Key words: CD97; class II seven-span transmembrane receptors; EGF-TM7 family; histology Acknowledgments: We thank Petra den Otter for expert technical assistance and Danny Dukers for computer editing of the immunohistological stainings. J. H. is a fellow of the Royal Netherlands Academy of Arts and Sciences.

Tissue distribution of the human CD97 EGF-TM7 receptor

Abstract: CD97 is a founding member of the EGF-TM7 family of class II seven-span transmembrane (7-TM) receptors. CD97 has an extended extracellular region with several N-terminal epidermal growth factor (EGF)-like domains, which mediate binding to CD55. Previous studies demonstrated the expression of CD97 on activated lymphocytes, monocytes, macrophages, granulocytes, and numerous haematopoietic and nonhaematopoietic cell lines. Here, we determined the cellular distribution of human CD97 in situ by immunohistochemistry (IH) and immunofluorescence (IF). Abundant expression of CD97 was detected on all types of macrophages and dendritic cells, except for microglia. Within the lymphoid lineage, most T cells but only a few B cells express CD97. Germinal centre B cells do not express the molecule. Except for smooth muscle cells, no staining was found on other cells outside the immune system. However, analysis of a restricted set of epithelial tumors revealed CD97 expression on the malignant cells in thyroid and gastrointestinal tract cancer.

Authors’ affiliations:

During the Fifth Workshop on Leukocyte Differentiation Antigens,

Correspondence to: Dr. Jörg Hamann Clinical Immunology Department AMC P.O. box 22700 1100 DE Amsterdam The Netherlands Tel: π31 20 5667687 Fax: π31 20 6091222 e-mail: j.hamann/amc.uva.nl

CD97 was defined as an activation-induced antigen on lymphocytes (1, 2). While present at low levels on resting cells, the molecule is upregulated within 1–4 h from activation on virtually all lymphocytes, where high expression persists for several days. Furthermore, CD97 is found on monocytes, granulocytes, and numerous hemato-

L.H. Jaspars1, W. Vos1, G. Aust2, R.A.W. van Lier3, J. Hamann3 1 Department of Pathology, Academic Hospital, Free University, Amsterdam, The Netherlands, 2

Institute for Pathology, Medical Faculty, University of Leipzig, Leipzig, Germany,

3

Department of Immunobiology, CLB and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

poietic and nonhematopoietic cell lines (1–5). In limited histological studies, CD97 expression was detected on spleen macrophages, alveolar macrophages, and Kupffer cells (6) and at high levels on leukocytes in some inflamed tissues (7). Molecular cloning of CD97 and the related EMR1 has defined a new group of class II seven-span transmembrane (7-TM) receptors designated EGF-TM7 family (7–12). EGF-TM7 family members are Received 17 November 2000, revised, accepted for publication 14 February 2001 Copyright c Munksgaard 2001 Tissue Antigens . ISSN 0001-2815 Tissue Antigens 2001: 57: 325–331 Printed in Denmark . All rights reserved

evolutionary related to the secretin receptor family (13). Different from this group of peptide hormone receptors, they have an extended extracellular region with a varying number of N-terminal epidermal growth factor (EGF)-like domains. Adhesion studies dem-

325

Jaspars et al : Tissue distribution of human CD97

onstrated that both lymphocytes and erythrocytes specifically bind

exposed to normal goat or rabbit serum for 20 min to prevent non-

to CD97 transfectants. The adhesive counterstructure has heen iden-

specific binding. Endogenous peroxidase was blocked by incubating

tified as CD55 or decay accelerating factor (DAF) (14), a molecule

the slides for 10 min with methanol/0.015% H2O2. Thereafter, the

involved in regulation of the complement cascade (15). Affinity of

slides were incubated with the mAb for 1 h at room temperature,

the CD55-binding site, which is formed by the EGF domain region,

then extensively washed in phosphate-buffered salt (PBS), incu-

differs between CD97 isoforms (16). The smallest isoform with three

bated with biotinylated F(ab)2 fragments of rabbit-anti-mouse Ig

EGF domains binds CD55 with significantly higher affinity than

(1:100; Dako) for 30 min, and washed again. Subsequently, strepta-

the larger isoforms containing four and five EGF domains. Recent

vidin-biotin complex conjugated to horseradish peroxidase (Strept-

characterization of mouse CD97 revealed a phylogenetic restriction

ABComplex, 1:100; Dako) was added for 1 h. The staining reaction

in the interaction between CD97 and CD55 (17, 18).

was performed in 0.5 mg/ml 3,3ø-diaminobenzidine tetrahydroxych-

Despite accumulating data on structure and molecular character-

loride (DAB) in PBS, pH 7.4, containing 0.01% H2O2 during 3–5

istics, the biological function of CD97 is still unknown. Since infor-

min. The slides were washed and counterstained with hematoxylin.

mation about the expression is necessary to understand the sup-

For doublestaining, the slides were incubated with the first anti-

posed role of CD97 in immunity, we have determined the in situ

body, which was demonstrated by the ABC method described

distribution in human. In addition, we analyzed where coexpression

above. Subsequently, the slides were incubated with the second anti-

of CD97 and CD55 is found in normal human tissues.

body for 1 h, which was visualized by an alkaline phosphatasemediated reaction as previously described (21). In this reaction, the second antibody was detected by incubating the slides respectively

Material and methods

with rabbit-anti-mouse Ig (1:50; Dako) and a monoclonal mouse alkaline-phosphatase-anti-alkaline-phosphatase

(APAAP)

complex

(1:50; Dako). Bound phosphatase was demonstrated with Naphtol

Antibodies

AS-MX as substrate and Fast Blue BB as coupling agent in 0.2 M The monoclonal antibodies (mAb) BL-Ac/F2 (anti-CD97, EGF do-

Tris buffer, pH 8.5 (30 min, at room temperature). The sections were

main 1), CLB-CD97/1 (anti-CD97, EGF domain 1), CLB-CD97/3 (anti-

then washed and mounted with glycerin gelatin.

CD97, stalk region), and CLB-CD97L/1 (anti-CD55) have been described previously (1, 14, 19). For double staining, the mAb O10

Immunofluorescence

(anti-CD1a; Immunotech, Marseille, France), Leu-4 (anti-CD3; Becton-Dickinson, San Jose, CA, USA), HD37 (anti-CD19; DAKO

For double IF staining, the following principle was applied. The

A/S, Glostrup, Denmark), 4C5 (anti-CD21; Dept. of Pathology, Free

slides were first incubated for 60 min with two primary mAb of

University, Amsterdam, The Netherlands), UCHL-1 (anti-CD45RO;

different Ig subclasses. Endogenous peroxidase was blocked by in-

kindly provided by Prof. P. Beverley, University College, London,

cubating the slides for 10 min with methanol/0.015% H2O2. Then,

UK), KP1 (anti-CD68; DAKO A/S), and AE1/3 (anti-cytokeratin;

a mixture of two goat-antimouse antibodies directed against the Ig

Boehringer, Mannheim, Germany) were used.

subtypes of the different primary antibodies (including 10% normal goat serum and normal human serum to prevent unspecific binding)

Tissues and fixation procedure

was added. One of these secondary antibodies was labeled with biotin, the other one was coupled to alkaline phosphatase (AP).

Normal tissues and tumors used in this study were derived of sur-

After incubation, the slides were first incubated with a streptavidi-

gically removed specimen. The tissues were snap frozen and stored

ne-biotin complex coupled to horse-radish peroxidase (sABC-HRP,

in liquid nitrogen. Frozen sections (4-mm thick) were cut, mounted

1:200; Dako) and than exposed to Cy-3 tyramide (1:200) with 0.03%

on poly-L-lysine-coated glass slides, and air-dried overnight. There-

H2O2 (22). The bound Cy-3 tyramide fluoresce bright red in the

after, they were fixed in acetone for 10 min and air-dried before

TRITC filter setting. The AP substrate-coupled antibody was visu-

performing IH.

alized by an enzyme-labeled fluorescence (ELF) method as recently described (23), resulting in a precipitate that fluoresce bright yellow-

Immunohistochemistry

green in the DAPI filter setting. The slides were than fixed with 4% paraformaldehyde for 10 min and counterstaned with DAPI (4 mg/

For IH, a modification of the biotin-streptavidin immunoperoxidase

ml). The sections were mounted with ELF mounting serum and

method (ABC method) (20) was used. The frozen sections were first

stored at 4æC.

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Tissue Antigens 2001: 57: 325–331


Results

Myeloid cells

Tissue distribution of CD97

In all tissues, CD97 mAb most intensively stained all types of macrophages and dendritic cells. In tonsils, lymph nodes, mucosa-associ-

To explore which cell types express CD97 in situ, a detailed IH and

ated lymphoid tissue (MALT), and the white pulp of the spleen, this

IF analysis on normal human tissues was performed using the mAb

includes starry sky macrophages, paracortical macrophages, interdi-

CLB-CD97/l, CLB-CD97/3, and BL-Ac/F2. No significant differences

gitating dendritic cells (IDC), and follicular dendritic cells (FDC), in

were found in the reactivity of the three mAb, which bind to differ-

lymph nodes as well sinusoidal histiocytes, and in the thymus cortical

ent regions of the CD97 receptor. The results of this analysis are

and medullary macrophages. Macrophages of the red pulp of the

depicted in Table 1 and Fig. 1.

spleen also showed strong expression of CD97 (Fig. 1C).

Table 1

Localization of CD97 in normal human tissues Cell type Tissue

Macrophages/ Histiocytic cells

Dendritic cells

Lymphoid cells 2

Other cell types1

Tonsil

π

π

π

ª

Lymph node

π

π

π2

ª

2

ª

Spleen

π

π

π

Thymus

π

π

ª

ª

Bladder

π

π

π

ª

Adrenal gland

π

np

np

ª

Parathyroid gland

np

np

np

ª

Intestine

π

np

np

ª

MALT

π

π

π2

ª

Heart

π

np

np

ª

Brain

π3

np

np

ª

4

Skin

π

π

np

ª

Liver

π5

np

np

ª

Lung

π

π

π

ª

MALT

π

π

π2

ª

Stomach

π

np

π

ª

MALT

π

π

π

ª

π

np

np

mesangial cells

Kidney Ovary

np

np

np

ª

Pancreas

np

np

np

ª

Prostate

π

np

π

ª

Thyroid gland

π

np

np

ª

Salivary gland

π

np

np

ª

Muscle

np

np

np

ª

Testis

π

np

np

ª

Uterus

π

np

np

ª

np: not present 1 staining of smooth muscle cells was regularly found (see Results) 2 variable expression on different Iymphoid cell types (see Results) 3 perivascular macrophages, no staining with glial cells 4 including Langerhans cells 5 Kupffer cells and periportal histiocytes


327


Fig. 1. Localization of CD97 in human tissues by IH and IF. A) Overview on the expression pattern of CD97 in tonsil. In the paracortex (P), cellular staining of lymphoid cells is observed (as is shown in more detail in D-E). In the lymphoid follicle (F), cell bodies of FDC are stained. B) In an area within tonsil, dominated by crypt epithelium (E), CD97 is most prominently expressed on intraepithelial and directly subepithelial located lymphoid cells. C) In spleen, CD97 is abundantly expressed on macrophages and sinus lining cells of the red pulp (RP) within the spleen (WP, white pulp). D) Double IF staining of tonsillar tissue for CD97 (red) and CD68 (green), showing that all CD68-positive cells also react with the CD97 mAb (resulting in a yellow staining of the cells). E) Overview and detail of double IF staining of tonsillar tissue for CD97 (red) and CD45RO (green), showing that in the paracortex (P) part of the lymphoid population doublestains (staining

328


yellow). The solely red staining cells are mostly macrophages. F) In the medulla of the thymus, CD97 expression is restricted to macrophages. Expression on epithelial cells was excluded by double staining for CD97 and cytokeratin (not shown). G) IF staining of tonsillar tissue depicts double expression of CD97 (red) and CD55 (green) on the FDC (F) in a lymphoid follicle in (yellow staining). H) Double IF staining of the epidermis (E) of the skin for CD97 (red) and CD1a (green), showing all Langerhans cells (arrows) being double positive (staining yellow). I) In kidney, CD97 is expressed on mesangial cells (arrow) within a glomerulus. J) In a follicular carcinoma of the thyroid gland, CD97 expression is most prominent on the poorest differentiated part of the tumor (asterisks). K) Expression of CD97 in an anaplastic thyroid carcinoma. L) Expression of CD97 on a poorly differentiated (singlet cell) carcinoma of the stomach.


In nonlymphoid tissues, different types of resident macrophages,

the mamma (all poorly differentiated), two adenocarcinomas of the

e.g. alveolar macrophages in the lung and Kupffer cells in the liver

stomach (moderately and poorly differentiated), three adenocarcin-

were markedly labeled by CD97 mAb. In the skin, applying double

omas of the large intestine (two moderately differentiated, one

staining for CD97 and CD1a, CD97 could be detected on Langerhans

poorly differentiated), two squamous cell carcinomas, and two aden-

cells within the epidermis and dermis (Fig. 1H). In the kidney, CD97

ocarcinomas of the lung (both moderately differentiated). If present,

expression was found on mesangial cells of the glomeruli (Fig. 1I).

macrophages and tumor-infiltrating lymphocytes (TIL) were

These cells are considered to be derived from monocytes (24).

strongly CD97 positive. Additionally, tumor cells of the anaplastic

Microglia in the brain did not stain with CD97 mAb.

carcinoma of the thyroid gland stained strongly for CD97 (Fig. 1K),

Double staining for CD97 and CD68 demonstrated that CD97 is

whereas tumor cells of the follicular thyroid gland carcinomas

expressed on all kind of macrophages and not on a selected popula-

showed variable, mostly moderate to slight staining (Fig. 1J). Aden-

tion of these cells (Fig. 1D). Expression on FDC was confirmed by

ocarcinomas of the stomach (Fig. 1L) and of the large intestine

double staining with a CD21 mAb.

showed variable staining, which was most strongly in the poorly differentiated cases. On tumor cells of the other tested carcinomas,

Lymphoid cells

CD97 staining was very weak or undetectable.

In lymphoid tissues (tonsils, lymph nodes, spleen, and the MALT

Association of CD97 with CD55

of stomach, gut, and lung), lymphoid cells stained with the CD97 mAb, albeit with variable intensity. In tonsils, the highest staining

Since CD55 has been demonstrated as ligand for CD97, the tissues

intensity was detected on intraepithelial or directly subepithelial

listed in Table 1 were also stained with a CD55 mAb. In all tested

localized lymphocytes (Fig. 1B). Weak staining for CD97 was found

tissues, CD55 expression was found on reticular cells, in particular

on lymphocytes of the paracortical area (Fig. 1A). Lymphoid cells

those localized around some of the vessels. In lymphoid tissues,

of the lymphoid follicle showed no detectable CD97 staining, except

CD55 was also detected on FDC, but not on other dendritic cell

for a few scattered cells (Fig. 1A). Double staining with mAb for

types. In the kidney, expression was detected in the mesangial ma-

CD3, CD19, and CD45RO confirmed that CD97 is mostly expressed

trix, which surrounds the mesangial cells of the glomeruli.

on T cells (Fig. 1E). CD97-positive B cells were detected in the epi-

In double staining experiments, areas with a close association

thelium and in the mantle zone. On germinal centre B cells, CD97

between CD55- and CD97-expressing cells were found. In lymphoid

was undetectable. The few cells found strongly positive within the

follicles, FDC stained positive for CD97 and CD55 (Fig. 1G). Within

germinal centre appeared to be mostly T cells. Lymph nodes, the

the FDC network, these cells are closely connected to all other ger-

white pulp of the spleen, and MALT, which have basically the same

minal center cells, thus also the CD97-positive T cells and macro-

structure of lymphoid tissue as tonsils, showed similar expression

phages. Related to vessels, CD55 was seen on perivascular reticular

patterns in lymphoid follicles and paracortical areas. In the thymus,

cells whereas CD97 was found on lymphocytes and monocytes

no detectable CD97 expression on lymphoid cells could be found

within the vascular lumina and on lymphoid cells and macrophages

(Fig. 1F).

around the vessels. However, CD97 was also expressed on lymphoid cells and macrophages that are not surrounded by CD55-expressing

Other cell types

cells.

Where present, smooth muscle cells were stained, e.g. around vessels or located in bladder, intestine, heart, skin, stomach, muscle, and uterus. Thyrocytes were weakly stained by CLB-CD97/3, but

Discussion

not by other CD97 mAb. Other tissue components were nonreactive. In the present study, expression of CD97 was examined in virtually Epithelial tumors

all normal human tissues by IH and IF. The results imply that CD97 exceeds its main function on macrophages, dendritic cells,

In addition to normal tissues, a series of epithelial tumor specimen

and activated lymphocytes. Except for glial cells in the brain, CD97

was analyzed for immunohistochemical reactivity with CD97 mAb,

was detected on all types of macrophages and dendritic cells, with-

including three carcinomas of the thyroid gland (two follicular carci-

out any restriction and without any clear differences in staining

nomas, one anaplastic/undifferentiated), three adenocarcinomas of

intensity. Expression of CD97 by lymphoid cells was found to be Tissue Antigens 2001: 57: 325–331

329


more restricted. Within activated lymphoid tissue, weak expression

strating the validity of earlier immunohistochemical observations

of CD97 was detected on most T cells but only a few intraepithelial

on CD97 that used mAb to the first EGF domain (6, 7). Furthermore,

localized B cells. Obviously, expression on lymphocytes in situ is

these data suggest that expression profiles of CD97 and EMR2 are

more closely regulated than suggested by in vitro experiments (1–4)

at least partially overlapping. Indeed, by RNA blot analysis and

that revealed rapid upregulation of CD97 on all lymphocytes during

reverse transcription-polymerase chain reaction (RT-PCR), EMR2

activation. The previous definition of CD97 as activation-induced

transcripts have been detected in granulocytes and monocytes/

antigen on lymphocytes, therefore, needs to be revisited as result of

macrophages but not in lymphocytes (28). Comparing in detail the

this study.

expression pattern of the molecular twins CD97 and EMR2 will

These data provide further evidence that expression of EGF-TM7

await the generation of specific mAb to EMR2.

family members is closely linked to the immune system. The other

In this study we also tested a restricted set of malignant epi-

family member whose tissue distribution has been analyzed is mouse

thelial tumors and found that in some of them CD97 was expressed

EMR1, also known as F4/80. This molecule (30% amino acid identity

on the malignant cells. This has been described earlier for thyroid

to CD97) has been studied extensively by Gordon and co-workers in

cancer, where expression intensity correlates negatively with the

order to describe the mononuclear phagocyte system of the mouse

differentiation grade of the tumor (5). Next to carcinomas of the

((25–27) and references therein). Different from CD97, in situ express-

thyroid, we demonstrated CD97 expression on poorly differentiated

ion of F4/80 is restricted to mature macrophages, including microglia.

tumor cells of adenocarcinomas of the gastrointestinal tract.

Expression is downregulated in T-cell-dependent areas as indicated by its absence from macrophages of the white pulp.

Association between the expression of CD97 and its ligand CD55 was detected within germinal centers and close to blood vessels.

Recently, EMR2 has been identified as a novel human EGF-TM7

Recently, we could demonstrate colocalization in the lining of syn-

family member (28). Surprisingly, the EGF domain region of EMR2

ovial tissue from rheumatoid arthritis patients, where CD97 is ex-

differs at only six out of 236 amino acids from that of CD97. Due

pressed on macrophage-like synoviocytes and CD55 on fibroblast-

to this striking similarity, mAb to the first EGF domain of CD97,

like synoviocytes (29). At the same site, increased levels of soluble

like BL-Ac/F2 and CLB-CD97/1, cross-react with EMR2 whereas

CD97 can be detected (7, 29). Based on the observed predominant

mAb to the stalk region of CD97 (46% amino acid identity to

expression of CD97 on macrophages, dendritic cells, and activated

EMR2), like CLB-CD97/2 and CLB-CD97/3, does not recognize

T cells, a more detailed analysis of the co-localization of both mol-

EMR2 (28). Therefore, we carefully compared the in situ reactivity

ecules should focus on the analysis of inflamed and neoplastic

of BL-Ac/F2, CLB-CD97/1, and CLB-CD97–3 in this study. No sig-

tissues as well as secondary lymphoid organs in ongoing immume

nificant differences between the three mAbs were detected, demon-

responses.

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