Cell Tissue Res (1992) 269:375-382

Cell Tissue Research 9 Springer-Verlag 1992

Distribution of the 2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues Soren K. Moestrup 1, Jorgen Gliemann 1, and Gorm Pallesen 2 1 Institute of Medical Biochemistry,Aarhus University, DK-8000 Aarhus C, Denmark 2 Laboratory of Immunohistology, Institute of Pathology, Aarhus University Hospital, DK-8000 Aarhus C, Denmark ReceivedJanuary 29, 1991 / Accepted May 19, 1992 Summary. The hepatic ~z-macroglobulin receptor (eiMR)/low density lipoprotein receptor-related protein (LRP) binds and endocytoses e2-macroglobulin-proteinase complexes in plasma. In addition, it binds lipoproteins, a novel 40 kDa protein, and complexes between plasminogen activators and plasminogen activator inhibitor type-l. This study shows, for the first time, the tissue distribution of ~2MR/LRP as determined by immunohistochemistry with specific monoclonal antibodies. The analysis revealed c~2MR/LRP-expression in a restricted spectrum of cell types, including neurons and astrocytes in the central nervous system, epithelial cells of the gastrointestinal tract, smooth muscle cells, fibroblasts, Leydig cells in testis, granulosa cells in ovary, and dendritic interstitial cells of kidney. Monocytederived cells displayed marked e2MR/LRP expression in the phagocytes of liver, lung and lymphoid tissues, but no or low expression in antigen-presenting cells including Langerhans' cells of the skin. The high abundance of ~2MR/LRP in certain cell types of most organs suggests two main routes for ~2MR/LRP-mediated ligand clearance: (1) systemic removal in liver of circulating ligands, and (2) non-hepatic interstitial removal in different organs, including the brain. Key words: e2-Macroglobulin receptor - Low-density lipoprotein receptor-related protein Tissue distribution Brain - Macrophages - Immunohistochemistry - Man

The c~2-macroglobulin (e2M) receptor (ezMR) is a large two-chain membrane protein that binds the proteinaseactivated form of the plasma protein e2M (Moestrup and Gliemann 1989; Jensen et al. 1989; Ashcom et al. 1990). ~2M is a potent proteinase-inhibitor containing a "bait" region, that is substrate for a wide variety of proteinases, e.g., trypsin, chymotrypsin, plasmin, neuCorrespondence to ." S.K. Moestrup

trophil elastase and fibroblast collagenase. Cleavage leads to the formation of stable c~2M:proteinase complexes and the appearance of hitherto concealed receptor-binding domains, one in each of the four 180-kDa subunits of ~2M (for a review, see Sottrup-Jensen 1989). Binding of e2M:proteinase complexes to ezMR is followed by endocytosis and degradation of the ligand in lysosomes (Gliemann and Davidsen 1986). Disappearance of e2M:proteinase in the blood occurs with a half time of about 2 rain in the rat and is mainly accounted for by uptake into hepatocytes (Davidsen et al. 1985). Receptors have also been demonstrated in fibroblasts (Van Leuven et al. 1979), monocytes/macrophages (Kaplan and Nielsen 1979; Petersen et al. 1987; Moestrup et al. 1990) and placental syncytiotrophoblasts (Jensen et al. 1988). Recently, analysis of amino-acid sequences of tryptic and cyanogen bromide fragments of purified placental c~2MR, performed independently by two groups (Strickland et al. 1991 ; Kristensen et al. 1991), has shown identity with the previously cloned (Herz etal. 1988) low density lipoprotein receptor-related protein (LRP) and ~2MR. This receptor (in the following, designated e2MR) is synthesized as a 4525 amino-acid single chain precursor molecule and is proteolytically cleaved to generate the membrane-spanning 601 amino-acid //-chain and the 3920 amino-acid a-chain (Herz et al. 1990). It binds a 40-kDa protein originally found in cr preparations (Jensen et al. 1989; Ashcom et al. 1990). This e2MR-associated protein is heparin-binding, inhibits ezM:proteinase binding to e2MR (Moestrup and Gliemann 1991) and modulates binding of lipoproteins (Herz et al. 1991). It has recently been cloned, sequenced and identified (Strickland et al. 1991) as the human homologue of a rat protein shown to be a Heymann nephritis antigen (Pietromonaco et al. 1990). ~2MR also binds//migrating very low density lipoproteins (Beisigel et al. 1989; Kowal et al. 1989; Herz et al. 1991) but its physiological relevance in lipoprotein metabolism remains uncertain (Brown et al. 1991; Van Dijk et al. 1991). Furthermore, cczMR is capable of binding the complex be-

376 tween p l a s m i n o g e n activator and p l a s m i n o g e n activator inhibitor type-1 (Nykja~r et al. 1992). In the present study, the tissue distribution o f e z M R has been defined by i m m u n o h i s t o l o g i c a l staining with newly developed m o n o c l o n a l antibodies. The analysis has revealed an a b u n d a n t e z M R expression in a restricted subset o f cell types previously n o t recognized as ~ z M R - b e a r i n g .

Materials and methods

Tissues Normal and neoplastic human tissues were retrieved from the frozen tissue bank at the Laboratory of Immunohistology, Aarhus Kommunehospitat. Tissues had been obtained fresh, snap frozen and stored at - 7 0 ~ C until processing. In a few instances, tissue was obtained from autopsy cases, since ct2MR proved resistant to postmortem degradation.

Production of monoclonal antibodies Monoclonal antibodies were produced by fusing mouse myeloma cells (NS-1) with spleen cells from a BALB/c mouse immunized with affinity-purified c~2MR as earlier described (Moestrup et al. 1990; Moestrup and Gliemann 1991). Three IgG1 antibodies (A2MR~-I, A2MRc~-2 and A2MR~-3)reacting with distinct epitopes in the c~2MR or-chain, and one IgG1 antibody (A2MRfl-1) against the ezMR fl-chain were used. The antibodies recognized ~2MR electroblotted from sodium dodecyl sulphate gels and the specificities of the antibodies were tested by immunoblot analysis of solubilized membranes from human liver and placenta (Moestrup et al. 1990). No cross-reactivity with other proteins was detected. In the present immunohistochemical analysis, A2MR~t-2

was used routinely. Positive reactions in cells not previously recognized as ct2MR bearing were controlled using A2MRc~-I, A2MRc~-3 and A2MR/M ; no differences were detected.

Imrnunohistological staining Cryostat sections of 7 Ixm thickness were fixed in acetone and chloroform and stained with monoclonal antibodies using a three stage immunoperoxidase method as previously described (Pallesen et al. 1984). Smears of blood and bone-marrow were stained using a standard alkaline phosphatase anti-alkaline phosphatase method (Cordell et al. 1984). All analyses were carried out in at least triplicates. Controls consisted of primary antibody replaced by buffer and antibodies of irrelevant specificities. Furthermore, tissues from brain, bone marrow, liver and duodenum were controlled with antibody inactivated by preincubation with a 20-fold molar excess of purified placental c~2MR.

Results The n o r m a l tissue distribution o f the e 2 M R , as defined by the labelling with m o n o c l o n a l a n t i b o d y A 2 M R e - 2 , is given in Table 1. The reactivity o f this a n t i b o d y was similar to that o f m o n o c l o n a l antibodies with other epitope specificities in the c~2MR ~- a n d / % c h a i n (data not shown). N o n e o f these antibodies were capable o f labelling c~2MR in routinely processed formalin-fixed and p a r a f f i n - e m b e d d e d tissues. As expected, strong labelling was seen in cell types k n o w n to express e 2 M R . These cells include hepatocytes (Fig. 1 a), m a c r o p h a g e s (Fig. 1 a, b), m o n o c y t e s (Fig. 2 a), fibroblasts, lipocytes and syncytiotrophoblasts. H o w ever, a b r o a d spectrum o f other cell types o f different

Table 1. Distribution of e2MR in normal tissues as defined by immunohistochemical labelling with monoclonal antibody A2MRct-2 Tissue

Cell types labelled

Cell types not labelled

Lymph node and tonsil

Macrophages of follicular centres, sinuses and epithelioid type

Spleen

Macrophages of splenic cords, otherwise as indicated under lymph nodes Cortical and medullary macrophages

Lymphoid cells, plasma cells, follicular dendritic cells, interdigitating cells, sinus-lining cells, endothelial cells of postcapillary venules, squamous epithelium Sinus-lining cells, otherwise as indicated under lymph nodes Epithelial cells including Hassal's bodies, cortical and medullary thymocytes, interdigitating cells Granulocytes and their precursors, thrombocytes, megakaryocytes, erythrocytes, Iymphocytes

Lymphatic-haematopoietic system

Thymus Blood and bone marrow

Monocytes and their precursors, reticulum cells, erythroblasts (immature)

Alimentary system Tongue, oesophagus Gastric mucosa (corpus, pylorus) Intestine, small and large Gall bladder Liver Pancreas (exocrine) Salivary glands

Gastric pit epithelium Surface epithelium and crypts of Lieberkfihn Columnar epithelium Hepatocytes, Kupffer cells

Squamous epithelium, gland epithelium Fundic and pyloric gland epithelium Brunner's glands Bile duct epithelium Acinar and duct epithelium Gland and duct epithelium including myoepithelial cells

377 Table 1. Continued

Tissue Urinary system Kidney

Cell types labelled

Cell types not labelled

Glomerular mesangial cells, interstitial dendritic cells

Glomerular epithelium and capillaries convoluted and excretory tubules Transitional epithelium

Leydig cells

Seminiferous tubules including Sertoli cells Epithelium Epithelium Epithelium Epithelium

Ureter and urinary bladder Male reproductive system

Testis Epididymis Vas deferens Seminal vesicle Prostata Female reproductive system Vagina Cervix Uterine endometrium Salpinx Ovary Respiratory system Larynx

Lung Skin and appendages Skin

Stromal cells Granulosa cells

Respiratory epithelium (weak), glandular epithelium Bronchial and alveolar epithelium (weak), alveolar macrophages

Fraction of Langerhans' cells

Female breast Endocrine system Thyroid gland Parathyroid gland Adrenal gland Pancreatic islets Nervous system Cerebrum

Cerebellum

Squamous epithelium, sebaceous glands, sweat gland epithelium Epithelium including myoepithelium

Follicular epithelium Epithelial cells (partial weak) Medullary cells (weak)

Neurons (strong), a fraction of protoplasmic and fibritlary astrocytes, basal membrane of capillaries Granular cells, Purkinje cells (weak), astrocytes (fraction) and basal membrane of capillaries

Nerves Other structures Muscle Fat tissue Connective tissue Chondroid tissue Blood vessels Macrophages Pleura Placenta

Squamous epithelium Ecto- and endocervical epithelium Gland epithelium Epithelium Ova

Cortical cells Islet cells

Oligodendrocytes, a fraction of astrocytes, endothelium of capillaries Bergmann cells Peripheral cells

Smooth muscle Lipocytes Fibroblasts Chondrocytes Tunica media (myofibrocytes), pericytes Most types (see Table 2) Mesothelial cells Syncytiotrophoblast, decidua cells, Hofbauer cells

Skeletal and cardiac

Endothelium

C~

379

Fig. 2 a, b. Indirect immunohistochemical staining with monoclonal antibody A2MRe-2 against the a2-macroglobulin receptor (e2MR) e-chain using an alkaline phosphatase anti-alkaline phosphatase method, a Peripheral blood smear, x 1800. A positive monocyte

is seen at the right, contrasting with unstained neutrophil, lymphocyte and red cells, b Bone-marrow smear, x 1800. Immature erythroblasts stain stronger than ortochrome erythroblasts

origin were also labelled with A 2 M R e - 2 . In the alimentary system, the c o l u m n a r epithelial cells o f the gastrointestinal tract were stained (Fig. I c). A similar, a l t h o u g h weaker, staining o f epithelial cells o f the respiratory system (larynx, lung) was also seen. Fig. 1 d shows the absence o f labelling when using A 2 M R e - 2 p r e a b s o r b e d to purified placental a 2 M R . In the kidney, p r o n o u n c e d labelling was f o u n d in the interstitial dendritic cells o f the medulla (Fig. I e). a 2 M R was also detected in the glomerular mesangial cells. N o staining o f tubuli or capillaries was observed. In the central nervous system (Fig. l f-h), strong e 2 M R reactivity was seen in neurons. In the cerebellar cortex (Fig. 1 f), a distinct reaction was observed in the molecular layer a n d between granular cells c o r r e s p o n d ing to the terminal regions. The granular cell s o m a t a also a p p e a r e d reactive, whereas the Purkinje cells showed only minimal reactivity. Some cortical neurons displayed cytoplasmic labelling in addition to the surface labelling. Reactivity also occurred in other cell types o f the central nervous system, such as a fraction o f fibrillary and p r o t o p l a s m i c astrocytes (Fig. i g), whereas the B e r g m a n n glia cells and oligodendrocytes, for example, were negative. Sections o f fetal brain were m o r e inten-

sively stained (Fig. 1 h) than those o f adult brain. N o reactivity was observed in the peripheral nerves. Finally, a 2 M R reactivity was revealed in a diversity o f other cells, including s m o o t h muscle cells, c h o n d r o cytes, Leydig cells in the testis, granulosa cells in the ovary, and, surprisingly, erythroblasts in the b o n e marr o w (Fig. 2b). Variable expression was f o u n d a m o n g the n o r m a l s u b p o p u l a t i o n s o f cells o f the m o n o c y t e - m a c r o p h a g e