Biochemical and immunohistochemical characteristics of CD62 and CD63 monoclonal antibodies. Expression of GMP-140 and LIMP-CD63 (CD63 antigen) in ...
Virchows Archiv B Cell Pathol (1991) 61:269-277
VirchowsAw.h/vB CellPmtudogy l~,g
Mot~lar
9 Springer-Verlag 1991
Biochemical and immunohistochemical characteristics of CD62 and CD63 monoclonal antibodies Expression of GMP-140 and LIMP-CD63 (CD63 antigen) in human lymphoid tissues Marcel J. Metzelaar 1, Henk-Jan Schuurman 2, Harry F.G. Heijnen 1, Jan J. Sixma 1, and H. Karel Nieuwenhuis ~ 1 Department of Haematology, 2 Internal Medicine and Pathology, University Hospital Utrecht Received June 26 / Accepted August 9, 1991
Summary. During platelet secretion granule membrane glycoproteins are translocated to the plasma membrane. We report here the biochemical and immunohistochemical characterization of a panel of platelet-secretion-specific, CD62 and CD63 monoclonal antibodies (MoAb), which we raised to thrombin-activated platelets. The CD62 MoAb identify the a-granule membrane protein GMP-140, also designated platelet activation-dependent granule external membrane protein (PADGEM). The number of epitopes on thrombin-activated platelets ranged from 15000 to 20000. The CD63 MoAb recognize a 30-60 kDalton integral membrane protein of lysosomes. Due to its distinct localization, we have designated the CD63 antigen lysosome integral membrane protein, CD63 (LIMP-CD63). The number of epitopes on thrombin-activated platelets ranged from 9000 to 11000. Expression of GMP-140, a member of the Selectin family (also referred as the LEC-CAM family) of adhesion molecules, and LIMP-CD63 was examined on human spleen, thymus and lymph node by immunohistochemistry. Both GMP-140 and LIMP-CD63 showed a wide distribution in lymphoid tissues; vascular endothelial cells and tissue compartments that were readily accessible to blood-borne components were uniformly positive for GMP-140 and LIMP-CD63. Furthermore, LIMP-CD63 was expressed in polymorphonuclear granulocytes and macrophages. Key words: Monoclonal antibodies - Activated platelets - Immunohistochemistry
Introduction Subendothelial tissues are exposed when a vessel wall is damaged. The interaction of platelets with the subendothelium results in the initiation of hemostasis by which Offprint requests to: M.J. Metzelaar, Department of Haematology, P.O. Box 85500, 3508 GA Utrecht, The Netherlands
platelets adhere to the vessel wall, are activated and secrete the content of their granules. These platelet functions are mediated by a variety of receptors, present on the plasma membrane and intracellular granule membranes, that interact with proteins in the vessel wall. With platelet activation, changes in platelet morphology and membrane composition occur. The membrane receptors for adhesive proteins are exposed initiating the activation process, platelets degranulate and secreted proteins bind to the cell surface (Plow and Ginsberg 1981 ; Phillips et al. 1980; Adelman et al. 1987). During secretion, the granule membranes fuse with the plasma membrane and the granule membrane glycoproteins are translocated to the plasma membrane (Berman et al. 1986; Stenberg etal. 1985; Nieuwenhuis etal. 1987; Metzelaar et al. 1991). Several monoclonal antibodies (MoAb) recognize activation-specific determinants on the platelet surface. The expression of a-granule membrane proteins on the plasma membrane can be monitored by CD62 MoAb specific for the granule membrane protein GMP-140, also designated the platelet activation-dependent granule external membrane protein (PADGEM) (Berman et al. 1986; Stenberg et al. 1985; Modderman 1989 a). Recently, it has been reported that rapid neutrophil adhesion to stimulated endothelial cells, and the interaction of activated platelets with neutrophils and monocytes is mediated by GMP-140 expressed on the plasma membrane (Larsen etal. 1989; Geng etal. 1990; Toothill et al. 1990). CD63 MoAb recognize a 30-60 kDalton lysosome integral membrane protein, that we described previously (Nieuwenhuis et al. 1987; Metzelaar et al. 1991; Modderman 1989b). The CD63 antigen is expressed on the plasma membrane after secretion of lysosomes. It is identical to the previously described melanoma-associated antigen ME491 (Metzelaar et al. 1991; Hotta et al. 1988). The ME491 antigen is abundant in malignant melanomas, nevocellular nevi, neuroendocrine tumors and adenocarcinomas (Atkinson et al. 1984, 1985; Ernst et al. 1986). No biological funtion for the CD63 antigen is known at present.
270
We report here the biochemical and immunohistochemical characteristics of the CD62 and CD63 MoAb that we raised against thrombin-activated platelets. Due to its distinct localization in the lysosomal membrane, we have designated the CD63 antigen, lysosome integral membrane protein, CD63 (LIMP-CD63). The expression of GMP-140 and LIMP-CD63 in human spleen, thymus and lymph node was defined by immunohistochemistry.
Methods Platelet preparation. Blood was collected from healthy volunteers. Freshly fixed platelets were prepared by collecting blood in 10 volumes of paraformaldehyde (PFA, 1% v/v, final concentration). The first 2 ml of blood were discarded. For preparation of thrombin-activated platelets, blood was collected in a 0.1 volume of 135 mM trisodium citrate. Platelet-rich plasma was obtained by centrifugation (10 min, 200 x g, room temperature). After adding 5 mM ethylenediamine tetra-acetic acid (EDTA), the platelets were isolated by centrifugation (10min, 1000• room temperature) and washed twice in phosphate-buffered saline (PBS) containing 5 mM EDTA. In some experiments, platelets were separated from plasma by gel-filtration on a Sepharose 2B (Pharmacia, Uppsala, Sweden) (Tangen etal. 1971) column, equilibrated in Ca2+-free Tyrode's solution, pH 7.25 containing 0.2% albumin.
3 volumes ofdibutylphtalaat) and centrifugation (3 min, 12000 x g, room temperature). The tube tips were cut off and the platelet bound radioactivity was determined in a gamma-counter. Nonspecific binding was determined by adding a 200-fold excess of unlabeled MoAb to the incubation mixture.
lmmunoprecipitations. Thrombin-activated platelets (2 x 109) were labeled with Na12SI using a modified lodogen procedure (Fraker and Speck 1978). The platelets were solubilized for 1 h at 4* C with 10 mM Tris, 150 mM NaCI buffer pH 7.8, containing 1% Nonidet P-40 (NP-40) and the protease inhibitors 2 mM phenylmethylsulphonyl fluoride (PMSF), 10mM D-aminobenzidine, 20 ~tg/ml Soybean trypsin inhibitor and 5 mM EDTA. The lysate was centrifugated (15 min, 10000 x g, 4~ C) to remove undissolved material and precleared with normal mouse IgG that had been covalently coupled to CNBr-Sepharose 4B (Pharmacia) according to the manufacturer's instructions. Purified IgG was coupled to CNBr-Sepharose 4B (1 mg/ml gel) and aliquots of the IgG-Sepharose beads were incubated with the cell lysate for 3 h at room temperature. The beads were washed twice with 1 M NaCI, 1% NP-40 pH 6.6, twice with 100 mM Tris, 1% NP-40 pH 7.4, twice with 20 mM Tris pH 7.4, and twice with 150 mM NaCI pH 7.4. The bound protein was extracted from the Sepharose beads by boiling for 10 min in electrophoresis sample buffer (125 mM Tris-HC1 pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.025% bromophenol blue with or without 5% ~-mercaptoethanol). The extracts were analyzed on a 3-25% SDS-polyacrylamide gradient gel. Subsequently, the gels were dried and autoradiography was performed. The molecular weight (MW) was determined using a mixture of low MW proteins (Pharmacia) and high MW proteins (Bio-Rad, Richmond, Va, USA).
Monoclonal antibody preparation. Platelet activation specific MoAb were produced by immunizing Balb/c mice intraperitoneally with 3 • 109 thrombin-activated PFA-fixed platelets. This was repeated 2 weeks later and a booster injection was given intraperitoneally with 5 • 109 PFA-fixed thrombin-activated platelets on day 21. After a further 3 days, spleen cells were fused with X63 Ag8.653 myeloma cells according to standard procedures (K6hler and Milstein 1976). Hybridoma selection was performed using an enzymelinked immunosorbent assay (ELISA); freshly fixed or thrombinacivated platelets were used as antigen. Hybridoma cells producing antibodies that bound to fixed thrombin-activated platelets, but not to freshyl fixed platelets, were subcloned twice by limiting dilution. The MoAb designated RUU-SP 1.18, 2.15 and 2.17 were clustered as CD62 at the I V th International Conference on Human Leucocyte Differentiation antigens (Metzelaar et al. 1989a; Modderman 1989a). The MoAb designated RUU-SP 2.19, 5.15 and 5d10 recognized a 30-60 kDalton protein. These MoAb mutually blocked RUU-SP 2.28 and were clustered as CD63 at the I V th International Conference on Human Leucocyte Differentiation antigens (Metzelaar et al. 1989b; Modderman 1989b).
Antibody purification and iodination. Monoclonal antibodies were purified from ascitic fluid using protein A-Sepharose (Pharmacia) according to the manufacturer's instructions. IgG fractions were pooled, dialyzed against distilled water, lyophilized and stored at -20~ until used. Purified lgG was radiolabeled with Na125I (Amersham, Buckinghamshire, UK) by a modified lodogen procedure (Fraker and Speck 1978). Non-covalently linked 125I was removed by separation on a G25-M column (Pharmacia) equilibrated with PBS. The specific activity ranged between 400 and 600 cpm/ ng. More than 98% of the radioactivity was precipitated by 10% trichloroacetic acid.
Western blot analys&. Cells were solubilized for 1 h at 4~ in 10 mM Tris, 150 mM NaC1 buffer pH 7.8, containing 1% Nonidet P-40 and the protease inhibitors 2 mM PMSF, 10 mM D-aminobenzamidine, 20~tg/ml Soybean trypsin inhibitor and 5mM EDTA. The lysate was centrifuged (15 min, 10000xg, 4~ C) to remove undissolved material, and 50 Ixg protein was analyzed on a 5-15% SDS-polyacrylamide gel. After transfer to an Immobilon membrane (Millipore, Bedford, USA), non-specific binding was blocked in PBS/5% non-fat dry milk (Protifar). Subsequently, the sheets were incubated with MoAb (1:1000 ascites in PBS/0.5% Protifar) for 1 h at room temperature. After washing three times with PBS/0.05% Tween-20, the filters were incubated with tzsIlabeled sheep-anti-mouse F(ab')2 (Amersham, specific activity 15 ItCi/lag, 1 ~tCi/mi in PBS/0.5% Protifar) for I h at room temperature. Unbound label was removed by washing and autoradiography was performed. lmmunoelectron microscopy. For immunoelectron microscopy, ultrathin cryosectioning and immunogold labeling according to Tokuyasu (1978) were performed as adapted by Slot et al. (1988). Briefly, gel-filtered resting or thrombin-activated platelets were fixed in 2% PFA, 0.2% glutaraldehyde in PBS for 2 h at 4~ C. After washing twice in PBS/0.15% glyeine the pellets were embedded in 10% gelatine. Ultrathin cryosections were cut and incubated with purified lgG from CD62 or CD63 MoAb for 30 min at room temperature. This was followed by a second incubation with rabbit anti-mouse immunoglobulins, and finally, the sections were labeled with 10 nm colloidal gold particles conjugated to protein-A. An non-specific antibody was used as a control and no labeling was observed with this.
Immunohistochemistry and enzyme histochemistry. This study was Binding of IZSl-antibody to platelets. Binding of 125I-MoAb to PFA-fixed resting platelets or washed thrombin-activated platelets was studied by incubating varying concentrations of MoAb with platelet suspensions (108 platelets/ml, final concentration) in a final volume of 120 lal. After incubation for 2 h at room temperature, platelets and unbound antibody were separated by layering 100 lal aliquots on top of 100 p.1 phtalate (2 volumes dinonylphtalaat,
performed on frozen specimens of human thymus, spleen and lymph node received by the Department of Pathology for histopathologic diagnosis. By conventional histology these showed a normal architecture and cellular composition. Cryostat sections (6 ~tm) were fixed in acetone for 10 min at room temperature and then incubated with the MoAb at predetermined optimal dilutions for 60 min. This was followed by a second incubation with rabbit
271
anti-mouse immunoglobulins conjugated with horseradish peroxidase, and a third incubation with swine anti-rabbit immunoglobulins conjugated with horseradish peroxidase (both conjugated antisera from Dakopatts, Glostrup, Denmark). Colour development was performed with 3-amino-9-ethyl carbazole (Aldrich Chemical Co., Milwaukee, Wis, USA) with H202 as the substrate. The sections were embedded in an aqueous solution of gelatin (I 8% w/v), glycerin (50% w/v) and phenol (1% w/v). Negative controls included omission of the MoAb in the first incubation or replacement of this MoAb by an irrelevant one. No specific labeling product was observed in these controls. In serial sections, enzyme histochemistry was performed for fl-glucuronidase, alkaline phosphatase, acid phosphatase, ATPase, and non-specific esterase activities following conventional procedures (Pearse 1985).
Platelet aggregation studies. Platelet aggregation studies were performed in an aggregometer at 37* C with 5 laM adenosine diphosphate (ADP), 5 p.M epinephrine, 1 mg/ml ristocetin or 4 lag/mlcollagen. The aggregation studies were performed after preincubation with 5-20 lal ascitic fluid at 37~ C for 30 min. In some experiments unstirred gel-filtered platelets were incubated simultaneously at room temperature with thrombin and MoAb. After 5 min, aggregation was initiated by stirring the platelets in a aggregometer at 37* C, adding 100 lag/mlfibrinogen and 100 laM CaCI2.
200-
uUm
94-
67-
43Results
Preparation of platelet activation-specific monoclonal antibodies Six platelet activation-specific MoAb were identified using an ELISA with either freshly fixed or fixed thrombin-activated platelets as antigen. The MoAb, designated RUU-SP 1.18, 2.15, 2.17, 2.19, 5.15 and 5d10, reacted preferentially with thrombin-activated platelets. All MoAb were of the IgG1 subclass. Purified IgG of the various MoAb did not cause platelet aggregation in the absence of an agonist, and did not inhibit platelet aggregation induced by ADP, thrombin, collagen and ristocetin. To investigate the effect of the MoAb on the platelet function during stimulation, G F P were incubated with purified IgG and thrombin simultaneously. After 5 min the aggregation reaction was initiated by adding fibrinogen and stirring. There was no effect on platelet aggregation induced by ADP, epinephrine, collagen, ristocetin and thrombin.
Characterization of the antigens recognized by the activation-specific monoclonal antibodies lmmunoprecipitation and Western blotting were used to characterize the molecular weight of the platelet proteins recognized by the various MoAb. The protein identified by RUU-SP 1.18, 2.15, 2.17 was immunoprecipitated from the lysate of 125I-ectolabeled thrombin-activated platelets and analyzed on a SDS-polyacrylamide gel. The MoAb precipitated a protein with a molecular weight of 140 kDalton under nonreducing, and of 150 kDalton under reducing conditions, which corresponded to the molecular weight of GMP-140, also designated PADG E M (Fig. 1). These MoAb were clustered as CD62 at
1 2 3 4 5 6 Fig. I. Identification of platelet antigens recognized by various monoclonal antibodies by immunoprecipitation. The antigens were immunoprecipitated from a NP-40 lysate of ~2~I-ectolabeled thrombin-activated platelets. Subsequently, the precipitates were analyzed on a 5--15% SDS-polyacrylamide gel and visualized by autoradiography. GMP-140 is precipitated under nonreducing (lanes I to 3) and reducing conditions (lanes 4 to 6). Lanes / and 4: RUU-SP 1.18, 2 and 5: RUU-SP 2.15, 3 and 6: RUU-SP 2.17
the IV th International Conference on Human Leucocyte Differentiation antigens (Metzelaar et al. 1989a; Modderman 1989a). With RUU-SP 2.19, 5.15 and 5d10, no radiolabeled proteins could be precipitated. The antigen recognized by RUU-SP 2.19, 5.15 and 5d10 was identified by Westen blot analysis of platelet proteins from solubilized resting platelets separated on a SDS-polyacrylamide gel (Fig. 2). Under nonreducing conditions, the heavily glycosylated CD63 antigen was identified with a molecular weight ranging from 3060 kDalton. RUU-SP2.28, which was clustered as CD63 at the IV th International Conference on Human Leucocyte Differentiation antigens (Metzelaar et al. 1989b; Modderman 1989b), and RUU-SP 2.15 are given for comparison.
s 25I-Antibody binding to resting and activated platelets Binding studies were performed with 125I-labeled IgG of the various MoAb to freshly fixed platelets or fixed thrombin-activated platelets. The binding of the MoAb to fixed thrombin-activated platelets was saturable. The binding curves were analyzed using Scatchard analysis (Fig. 3). This revealed a single class of binding sites on
272 Table 1. Number of binding sites and dissociation constant (Kd) of CD62 and CD63 MoAb on resting and thrombin activated platelets determined by Scatchard plot analysis MoAb RUU-SP
Resting platelets
Stimulated platelets
CD62
1.18 2.15 2.17
< 1000 < 1000 < 1000
17.150 19.500 15.050
3.7 5.1 4.2
CD63
2.19 5.15 5d10
< 1000 < 1000 < 1000
10.200 9.800 11.350
15.0 7.4 13.3
180-
O 116-
Kd (nM)
84-
58-
4g-
3626-
1
2
3
4
5
Fig. 2. Western blot analysis of platelet antigens recognized by various monoclonal antibodies. Solubilized resting platelets were run on a 5-15% SDS-polyacrylamide gel under nonreducing conditions and blotted on an Immobilon membrane. Blots were incubated with MoAb and a 125I-labeled sheep anti-mouse second antibody. The antigens were visualized by autoradiography. 30-60 kDalton lysosome integral membrane protein CD63 recognized by lane 1: RUU-SP 2.28, 2: MoAb RUU-SP 2.19, 3: RUU-SP 5.15, and 4: RUU-SP 5d10. Lane 5 : G M P - 1 4 0 identified by RUU-SP 2.15
0.8
thrombin-activated platelets. Freshly fixed platelets bound the MoAb minimally, the number of binding sites ranging from 600 to 1000 for the various CD62 and CD63 MoAb. On thrombin activated platelets 1500020000 binding sites were observed for the CD62 MoAb, and 9000-11000 for the CD63 MoAb. The dissociation constant (Kd) ranged from 3.7 to 5.1 and from 7.4 to 15.0 nM for the CD62 and CD63 MoAb respectively (Table 1). We performed cross-blocking studies to investigate whether the various MoAb recognize the same or different epitopes. In these experiments the binding of l zsI-labeled IgG of the various CD62 or CD63 MoAb to thrombin-activated platelets was blocked with an excess of non-labeled IgG from the same or other MoAb (Table2). The CD62 MoAb RUU-SP2.15 and 2.17 showed mutual blocking of binding, but no blocking with RUU-SP 1.18 was observed. The CD63 MoAb RUU-SP 2.28, 2.19, 5.15 and 5d10 mutally blocked each others binding.
Immunocytochemical localization of GMP-140 and LIMP-CD63 Immunoelectron microscopic studies were performed on cryosections of gel-filtered resting platelets. Using MoAb
0.4
A 0.6
B
9
0.3
0.4
0.2
o 0.2 m
0.1
0.0
9
1
2
3
'
4
0.0 0.0
0.5
1.0
1.5
2.0
2.5
BOUND (nld//lO 7 PLATELETS)
Fig. 3A, B. Scatchard analysis of 125I-labeled MoAb binding to paraformaldehyde-fixed, thrombin-activated platelets. Varying amounts of 125I-MoAb were incubated with thrombin (5 U/ml) activated platelets. After 2 h incubation at room temperature, platelets and unbound label were separated. Platelet bound radioactiv-
ity was measured and a Scatchard plot was calculated of the specific binding data. A CD62 MoAb RUU-SP 1.18 (o), RUU-SP2.15 (A), RUU-SP 2.17 (e). B CD63 MoAb RUU-SP 2.19 (o), RUUSP 5.15 (zx), RUU-SP 5d10 (A)
273 Fable 2. Percentage of specific binding to thrombin activated plate-
ets in cross-blocking studies of CD62 and CD63 MoAb. The bindng of t2SI-labeled IgG of the various CD62 and CD63 MoAb o thrombin-activated platelets was blocked with an excess nonabeled IgG from the same or other MoAb. The CD62 MoAb RUU-SP 2.15 and 2.17 showed mutual blocking of binding but ao blocking with RUU-SP 1.18 was observed. The CD63 MoAb RUU-SP 2.28, 5.15 and 5d10 mutually blocked each others binding
R U U - S P 1.18, 2.15 and 2.17, the label was found in the membrane of a-granules, confirming that the MoAb recognize the GMP-140. In Fig. 4 A the results are shown for MoAb R U U - S P 2 . 1 5 . Using the MoAb R U U SP 2.19, 5.15 and 5d10, gold labeling was restricted to the membrane of lysosomes. In Fig. 4B the results are shown for MoAb R U U - S P 2.19.
ED62 t25I.MoAb
Expression of GMP-140 and LIMP-CD63 in thymus, lymph node and spleen
Excess non-labeled MoAb
1.18 2.15 2.17
1.18
2.15
2.17
0 75 81
84 0 8
83 0 0
CD63 lzSI-MoAb
2.28 2.19 5.15 5d10
Excess non-labeledMoAb 2.28
2.19
5.15
5d10
0 4 2 0
1 0 3 0
2 2 0 1
1 3 1 1
Fig. 4A, B. Immunogold labeling of ultrathin cryosections of resting gel-filtered platelets with CD62 and CD63 MoAb. A CD62 MoAb RUU-SP 2.15 with 10 nm protein-A gold particles. The la-
CD62 MoAb R U U - S P 1.18, 2.15 and 2.17, directed against GMP-140, diffusely labeled the red pulp in sections of spleen. In the white pulp a dendritic staining of reticulum cells was observed, especially for MoAb 1.18. This labeling was most pronounced in the vascular reticulum of the follicles and pericytic cells surrounding the central arteriole (Fig. 5 A). The MoAb 2.15 and 2.17 showed a less intense labeling of the white pulp than MoAb 1.18. In lymph node sections, MoAb 1.18 also gave much stronger labeling than the MoAb 2.15 and 2.17. In lymph n o d e s , the blood vessels were labeled both in the medulla (Fig. 5 B) and cortex (Fig. 5 C). In addition, reticular components were stained, especially in the follicles. The labeling of blood vessels paralleled the enzyme activity for alkaline phosphatase. In sections
bel is restricted to the membrane of a-granules. B CD63 MoAb RUU-SP 2.29 with 10 nm protein-A gold particles. The label is restricted to the membrane of lysosomes. Bar represents 200 nm
Virchows Archiv B Cell Pathol (1991) 61:269-277
VirchowsAw.h/vB CellPmtudogy l~,g
Mot~lar
9 Springer-Verlag 1991
Biochemical and immunohistochemical characteristics of CD62 and CD63 monoclonal antibodies Expression of GMP-140 and LIMP-CD63 (CD63 antigen) in human lymphoid tissues Marcel J. Metzelaar 1, Henk-Jan Schuurman 2, Harry F.G. Heijnen 1, Jan J. Sixma 1, and H. Karel Nieuwenhuis ~ 1 Department of Haematology, 2 Internal Medicine and Pathology, University Hospital Utrecht Received June 26 / Accepted August 9, 1991
Summary. During platelet secretion granule membrane glycoproteins are translocated to the plasma membrane. We report here the biochemical and immunohistochemical characterization of a panel of platelet-secretion-specific, CD62 and CD63 monoclonal antibodies (MoAb), which we raised to thrombin-activated platelets. The CD62 MoAb identify the a-granule membrane protein GMP-140, also designated platelet activation-dependent granule external membrane protein (PADGEM). The number of epitopes on thrombin-activated platelets ranged from 15000 to 20000. The CD63 MoAb recognize a 30-60 kDalton integral membrane protein of lysosomes. Due to its distinct localization, we have designated the CD63 antigen lysosome integral membrane protein, CD63 (LIMP-CD63). The number of epitopes on thrombin-activated platelets ranged from 9000 to 11000. Expression of GMP-140, a member of the Selectin family (also referred as the LEC-CAM family) of adhesion molecules, and LIMP-CD63 was examined on human spleen, thymus and lymph node by immunohistochemistry. Both GMP-140 and LIMP-CD63 showed a wide distribution in lymphoid tissues; vascular endothelial cells and tissue compartments that were readily accessible to blood-borne components were uniformly positive for GMP-140 and LIMP-CD63. Furthermore, LIMP-CD63 was expressed in polymorphonuclear granulocytes and macrophages. Key words: Monoclonal antibodies - Activated platelets - Immunohistochemistry
Introduction Subendothelial tissues are exposed when a vessel wall is damaged. The interaction of platelets with the subendothelium results in the initiation of hemostasis by which Offprint requests to: M.J. Metzelaar, Department of Haematology, P.O. Box 85500, 3508 GA Utrecht, The Netherlands
platelets adhere to the vessel wall, are activated and secrete the content of their granules. These platelet functions are mediated by a variety of receptors, present on the plasma membrane and intracellular granule membranes, that interact with proteins in the vessel wall. With platelet activation, changes in platelet morphology and membrane composition occur. The membrane receptors for adhesive proteins are exposed initiating the activation process, platelets degranulate and secreted proteins bind to the cell surface (Plow and Ginsberg 1981 ; Phillips et al. 1980; Adelman et al. 1987). During secretion, the granule membranes fuse with the plasma membrane and the granule membrane glycoproteins are translocated to the plasma membrane (Berman et al. 1986; Stenberg etal. 1985; Nieuwenhuis etal. 1987; Metzelaar et al. 1991). Several monoclonal antibodies (MoAb) recognize activation-specific determinants on the platelet surface. The expression of a-granule membrane proteins on the plasma membrane can be monitored by CD62 MoAb specific for the granule membrane protein GMP-140, also designated the platelet activation-dependent granule external membrane protein (PADGEM) (Berman et al. 1986; Stenberg et al. 1985; Modderman 1989 a). Recently, it has been reported that rapid neutrophil adhesion to stimulated endothelial cells, and the interaction of activated platelets with neutrophils and monocytes is mediated by GMP-140 expressed on the plasma membrane (Larsen etal. 1989; Geng etal. 1990; Toothill et al. 1990). CD63 MoAb recognize a 30-60 kDalton lysosome integral membrane protein, that we described previously (Nieuwenhuis et al. 1987; Metzelaar et al. 1991; Modderman 1989b). The CD63 antigen is expressed on the plasma membrane after secretion of lysosomes. It is identical to the previously described melanoma-associated antigen ME491 (Metzelaar et al. 1991; Hotta et al. 1988). The ME491 antigen is abundant in malignant melanomas, nevocellular nevi, neuroendocrine tumors and adenocarcinomas (Atkinson et al. 1984, 1985; Ernst et al. 1986). No biological funtion for the CD63 antigen is known at present.
275 of thymus, the CD62 MoAb to GMP-140 labeled only the connective tissue between the thymic lobules and blood vessels (data not illustrated). CD63 MoAb RUU-SP 2.19, 5.15 and 5d10, directed against LIMP-CD63, labeled sections of spleen in a similar pattern to that obtained with RUU-SPI.18 (Fig. 5 D). The most pronounced staining was observed in cells immediately surrounding the central arteriole in the white pulp, and uniformly in ceils in the red pulp. This labeling paralleled the alkaline phosphatase enzyme activity (data not illustrated). In sections of lymph (illustrated for the medulla in Fig. 5E) strong labeling of blood vessels (including high endothelial venules in the paracortex) was observed in a pattern resembling that seen for alkaline phosphatase activity. In addition, there was faint positivity of the reticulum and of polymorphonuclear granulocytes and macrophages, especially in medullary cords. In sections of thymus (Fig. 5 F) staining of the connective tissue was observed. The capsule and solitary cells in the connective tissue, presumably polymorphonuclear granulocytes, were strongly labeled. The cortex was almost negative except for faint labeling of blood vessels. The medulla showed a low intensity reticular staining. Pronounced labeling was seen in blood vessels and cells in perivascular areas (presumably polymorphonuclear granulocytes and macrophages).
Discussion We report the biochemical and immunohistochemical characterization of a panel of CD62 and CD63 MoAb raised against activated platelets. Using these MoAb, GMP-140 and LIMP-CD63 (CD63 antigen) expression in the human thymus, spleen and lymph node has been defined in an immunohistochemical study. Biochemical characterization revealed that MoAb RUU-SP 1.18, 2.15 and 2.17 recognize the 0t-granule membrane protein GMP-140. At the IV th International Conference on Human Leucocyte Differentiation antigens these MoAb were clustered as CD62 (Metzelaar et al. 1989a; Modderman 1989a). MoAb RUU-SP 2.19, 5.15 and 5d10 identify a 30-60 kDalton protein. Cross-
Fig. 5. Immunohistochemistry. Labeling of spleen (A white pulp and surrounding red pulp) and lymph node (B medullary area and C cortex) by MoAb RUU-SP 1.18 directed to GMP-140. Note the labeling of cells surrounding the central arteriole in spleen (arrow) and blood vessels in lymph node (arrows). In addition, the labeling of connective tissue in the lymph node tissue section is shown. Labeling of spleen (D white pulp and surrounding red pulp) and lymph node (E medullary area) by MoAb RUU-SP5.15 (LIMP-CD63). Note the labeling of cells surrounding the central arteriole in spleen (arrow), and blood vessels and macrophage-like cells in medullary cords of the lymph node (arrows). Labeling of thymus (F) by MoAb RUU-SdI0 (LIMP-CD63). Note the strong labeling of the capsule, solitary cells in the connective tissue surrounding the thymic lobules (arrow), and blood vessels and cells in perivascular areas, especially in the cortico-medullary junction (arrowheads)
276
blocking studies with CD63 MoAb RUU-SP 2.28 (Metzelaar et al. 1989b; Modderman 1989b) revealed that this was the CD63 antigen. Based on the CD63 gene structure and recent electron microscopic studies (Metzelaar et al. 1991), we identified the CD63 antigen as an integral membrane glycoprotein of lysosomes. Due to this distinct localization, we have designated the CD63 antigen lysosome integral membrane protein CD63 (LIMP-CD63). Both CD62 and CD63 MoAb react preferentially with activated platelets and represent a marker for the platelet a-granule membrane and lysosome membrane fusion with the plasma membrane respectively. In cross-blocking experiments, the CD62 MoAb RUU-SP 2.15 and 2.17 showed mutual blocking of binding, but no blocking with RUU-SP 1.18 was observed. This indicates that RUU-SP 2.15 and 2.17 are directed against identical or partially overlapping epitopes, and that RUU-SP 1.18 recognizes a different epitope. The difference between RUU-SP 1.18 on the one hand, and RUU-SP 2.15 and 2.17 on the other, is reflected in the immunohistochemical characterization, where the MoAb RUU-SP 2.15 and 2.17 showed a less intense labeling than RUU-SP 1.18. This phenomenon may be ascribed to the labeling of an epitope in the sections by RUU-SP 1.18 that is better accessible for the antibody than the epitopes labeled by RUU-SP 2.15 and 2.17. The CD63 MoAb RUU-SP 2.28, 2.19, 5.15 and 5d10 mutally blocked each others binding, suggesting that they all recognize similar epitopes. However, we cannot exclude that these results were partially due to steric hindrance by IgG molecules already bound to an epitope in the blocking experiments. In particular, the distinct epitopes on the 30-60 kDalton LIMP-CD63 could easily be shielded by the much larger 150 kDalton IgG molecules. GMP-140 is a member of the newly defined family of Selectins (Marx 1989) (also referred to as the LECCAM family by Brandly et al. (1990)), which are inducible receptors on vascular cells (Marx 1989). This family, consisting of the endothelial leucocyte adhesion molecule ELAM-1, the lymphocyte homing receptor MEL14, and GMP-140, participates in cell-cell interactions between leucocytes and their target tissues (Bevilacqua et al. 1989). Recently, it has been reported that rapid neutrophil adhesion to stimulated endothelial cells and the interaction of activated platelets with neutrophils and monocytes is mediated by GMP-140 expressed on the plasma membrane of platelets and endothelial cells (Larsen etal. 1989; Geng etal. 1990; Toothill etal. 1990). The expression of GMP-140 was observed in platelets, megakaryocytes and endothelial cells (Stenberg et al. 1985; Beckstead et al. 1986; McEver et al. 1987; Bonfanti et al. 1989). Such a restricted expression was not observed by immunohistochemistry in lymphoid tissues. The tissue compartments readily accessible to blood-borne components, in particular, the red pulp of the spleen and, to a lesser extent, the lymph node medulla were uniformly positive for GMP-140, while those areas not readily accessible to the blood such as the
thymic cortex, which is protected by the thymus-blood barrier, show almost no labeling. The thymic medulla, which is not similarly protected does, however, stain with GMP-140. Vascular endothelium was almost uniformly positive in patterns indicating GMP-140 expression by the endothelial cells. This was substantiated by the co-expression of alkaline-phosphatase activity, commonly used to identify vascular endothelium, in serial sections. However, it cannot be excluded that the labeling observed is in part due to activated platelets bound to the endothelial cells in-situ. However, the cytoplasmic staining of endothelial cells argues against this mechanism as the sole explanation. We therefore concluded that almost all the endothelia in lymphoid organs express GMP-140 and that no special subsets of endothelial cells could be discriminated. Another remarkable feature was the strong labeling of cells immediately surrounding the endothelium of the central arteriole in the spleen white pulp. This labeling was more intense than that of the endothelium itself. It is tempting to suggest that pericytic histiocytic or fibroblastic reticulum cells at this location express GMP140, but the relevance of this remains to be established. On the other hand GMP-140 from blood-borne platelets may be trapped at this site and so explain this observation. That both GMP-140 and LIMP-CD63 expression was found at this location may argue in favour of this interpretation. LIMP-CD63 is a lysosomal membrane protein found in a wide variety of cells including platelets, macrophages and granulocytes (Nieuwenhuis et al. 1987; Modderman 1989b; Metzelaar et al. 1991). As with GMP140, a strong expression of LIMP-CD63 was observed in areas of lymphoid tissue readily accessible to bloodborne components and in vascular endothelial cells. Furthermore, polymorphonuclear granulocytes and macrophages were stained in the spleen, lymph node and thymus. We recently reported that LIMP-CD63 is identical to ME491, an antigen associated with human malignant melanoma (Metzelaar et al. 1991 ; Hotta et al. 1988). The ME491 antigen is abundant in malignant melanomas, nevocellular nevi, neuroendocrine tumors and adenocarcinomas (Atkinson et al. 1984, 1985; Ernst et al. 1986). However, we have observed a broad reactivity with normal tissues in the spleen, lymph node and thymus. This is confirmed by the observation that no tissue-specific LIMP-CD63 messenger RNA expression was observed in Northern blot analysis of various normal and transformed cells (Metzelaar et al. 1991).
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