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(Journal of Leukocyte Biology. 2000;68:650-654.)
© 2000 by Society for Leukocyte Biology

Clustering on the forward surfaces of migrating neutrophils of a novel GPI-anchored protein that may regulate neutrophil adherence and migration

Yukiko Nakamura-Sato^*, Katsunori Sasaki, Hiroshi Watanabe, Yoshihiko Araki^* and Fujiro Sendo^*

^* Department of Immunology and Parasitology, Yamagata University, School of Medicine;
Department of Anatomy, Shinshu University, School of Medicine, Matsumoto; and
Department of Nursing, Yamagata University, School of Medicine, Yamagata, Japan

Correspondence: Fujiro Sendo, Department of Immunology and Parasitology, Yamagata University, School of Medicine, 2-2-2, Iida-Nishi, Yamagata 990-9585, Japan. E-mail: fsendo{at}med.id.yamagata-u.ac.jp

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We previously reported a novel glycosylphosphatidylinositol (GPI)-anchored glycoprotein (tentatively designated GPI-80) on human leukocytes that may be involved in the regulation of neutrophil adherence and migration. In this study, we examined by immuno- and scanning electron microscopy, the distribution of GPI-80 on neutrophil surfaces. GPI-80 was diffusely distributed on the surface of resting neutrophils and on the peripheral areas of adherent cells after stimulation with N-formyl-methionyl-leucyl-phenylalanine. After longer stimulation (60 min), GPI-80 decreased in number and was again diffusely distributed on the surfaces of round neutrophils. Few GPI-80 were detected on the ventral surfaces of adherent neutrophils. Clusters of GPI-80 were detected on the forward surfaces of neutrophils transmigrating through pores of nitrocellulose membranes. These results may give a morphological background of possible role of GPI-80 for neutrophil extravasation.

Key Words: integrin • electron microscopy • localization

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Extravasation of peripheral blood leukocytes is an essential process that enables the cells to function in inflammatory and immune responses. However, little is known about the mechanisms that involve rapid cycles of leukocyte adhesion to and de-adhesion from the endothelium concurrent with their locomotion through the endothelium. To study this problem, we developed a mAb, designated 3H9, by screening for inhibition of human neutrophil adherence to plastic plates [¹ ]. This mAb enhances early phase ß₂ integrin-dependent neutrophil adherence and inhibits the late-phase responses, and also has similar effects on in vitro transendothelial migration of neutrophils [² ]. Furthermore, in the phagokinetic track assay, 3H9 induces neutrophil locomotion [³ ]. We performed molecular cloning of the molecule reacting with 3H9 and showed that it is a novel glycosylphosphatidylinositol (GPI)-anchored glycoprotein (tentatively designated GPI-80) that may modulate ß₂ integrin-dependent neutrophil adhesion and transendothelial migration [² ].

It is interesting that GPI-80 is highly homologous with Vanin-1, which has been reported to be localized in perivascular tissue of the mouse thymus and to be involved in prethymic cell homing to the thymus [⁴ ]. Both GPI-80 and Vanin-1 have approximately 40% homology with human biotinidase [⁵ ], suggesting that there is a superfamily of biotinidases that may be involved in leukocyte trafficking.

In the previous study using a confocal fluorescence microscopy, we found that (1) GPI-80 was localized on pseudopodia and adherent portions of polarized neutrophils, (2) that it was associated with CD11b/CD18 in resting neutrophils and moved to lamellipodia upon activation of these cells while CD11b/CD18 became localized on uropods, and (3) that this dissociation is dependent on extracellular calcium [Sendo, unpublished results].

In this study, we tried to obtain morphological information relating our previous experimental results on GPI-80 and we used backscatter electron images obtained by high-resolution, field emission scanning electron microscopy (FESEM). We examined the topographical distribution of GPI-80 on the surface of human neutrophils.

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Culture media and reagents
Cells were incubated in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS; ICN Biomedicals, Osaka, Japan). The reagents used were purchased from the following companies: N-formyl-methionyl-leucyl-phenylalanine (fMLP) and fibrinogen, Sigma Chemical, St. Louis, MO; Ficoll-Paque, Pharmacia Fine Chemicals, Uppsala, Sweden; heparin sodium injection-N, Shimizu Pharmaceutical, Shimizu, Japan; Dextran 200, ethanol, butyl alcohol, WAKO Pure Chemical, Osaka, Japan; sodium cacodylate, TAAB, Berks, UK; and glutaraldehyde, Nissin EM, Tokyo, Japan. University of Wisconsin (UW) solution (100 mM lactobionate, 30 mM raffinose, 25 mM KH₂PO₄, 5 mM MgSO₄, 3 mM glutathione, 5 mM adenosine, 1 mM allopurinol, 105 mM KOH, 20 mM NaOH), which had been introduced in organ preservation for transplantation in 1986 [⁶ ], was used in the preparation of tissues for electron microscopy to suppress swelling and bleb formation of cells [⁷ , ⁸ ]. All other reagents were of the highest grade commercially available.

Antibodies
Our previously described anti-GPI-80 monoclonal antibody (mAb), 3H9 (IgG1) [¹ ], was used in this study. A mAb, TCY-3, against Trypanosoma cruzi antigen, which does not cross-react with human neutrophils was used as an IgG1 control mAb. In the first experiments shown in Figure 1 , we used 15-nm colloidal gold particles conjugated with sheep anti-mouse IgG purchased from E-Y Laboratories (SanMateo, CA). In later experiments we used 10-nm particles (Funakoshi, Tokyo, Japan) because we found that labeling with 10-nm colloidal gold particles was more efficient than with 15-nm ones.

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Figure 1. Localization of GPI-80 on the apical cell surface. (A–C) low magnification of neutrophils observed by BSE imaging. Immediately after stimulation by fMLP, neutrophils had a round shape and random distribution of GPI-80 on the cell surface (A, 0 min). In the early phase of fMLP activation, cells displayed spreading margins on the substratum, and GPI-80 accumulation on their peripheral surfaces (arrows, B, 15 min). At 90 min of incubation, cells had a round shape with few projections on their surfaces, and the distribution of GPI-80 was diffuse and decreased in number (C, 90 min). (D–I) At high magnification, GPI-80 was distributed diffusely in some parts of the specimen (E), but in other parts in a clustered form (G and I, arrowheads). (D, F, and H) SE images. (E, G, and I) backscatter images of D, F, and H, respectively. Bars, 1.0 µm (A–C), 0.3 µm (D and E), and 0.1 µm (F–I).

Isolation of human neutrophils
Heparinized venous blood obtained from healthy volunteers was mixed with one-quarter volume of phosphate-buffered saline (PBS; pH 7.4) containing 3% Dextran 200 and allowed to stand at room temperature for approximately 60 min for erythrocytes to sediment. The leukocyte-rich supernatant was centrifuged at 400 g for 5 min at room temperature, the pellet was washed with PBS, and centrifuged on Ficoll-Paque density gradient at 400 g for 30 min at room temperature. The granulocyte-rich erythrocyte layer was treated with hypotonic shock and centrifuged at 300 g for 5 min. The pellet was collected and used as a source of neutrophils. It showed more than 96% purity by Giemsa staining.

Electron microscopy
For scanning electron microscopy (SEM) to examine the immunolocalization of GPI-80 on the apical surface of neutrophils, cells were seeded on glass coverslips in a 48-well flat-bottom culture plate (Falcon). For the observation of the ventral surface, cells were seeded onto a combination of two attached membranes. The upper ones were 10 µm thick PC MEMB 110615 membranes having multiple holes 10 µm in diameter (Nomura Micro-Science, Kanagawa, Japan) and the lower ones (PC MEMB 110606) had no holes. The lower membrane was incubated with 0.2% fibrinogen in the plate (Falcon) to coat the surface with fibrinogen at 37°C for 2 h. After washing three times with PBS, the upper membrane was overlaid on the treated lower membrane, and they were again treated with fibrinogen at 37°C for 2 h and then washed with PBS. After addition of 2 x 10⁶/mL neutrophils, the plates were centrifuged (200 g) for 3 min at 4°C and incubated at 37°C for 15 min in 5% CO₂ in air. We chose 15 min of incubation because many neutrophils adhered at this time. After the incubation, the two membranes were separated and the upper one was put upside down. Both membranes were washed twice with UW solution and incubated with 1% FBS/UW for 5 min at room temperature. We used the Boyden chamber technique [⁹ ], as modified by Zigmond and Hirsch [¹⁰ ], to observe the ventral surface of migrating neutrophils. The two compartments of the chamber (Nomura Micro-Science) were separated by a cellulose acetate filter with a pore size of 8 µm (Poretics, Livermore, CA). Neutrophils (2 x 10⁶/mL, 200 µL) in the upper chamber were incubated for 15 min at 37°C, with 10^-6 M fMLP in the lower compartment, and then the filters were stripped off. After blocking, the samples were incubated with 100 µL of 3H9 (10 µg/mL) for 30 min at 4°C and were then washed twice with UW solution. They were labeled with immunogold by incubation at 4°C with colloidal gold conjugated with goat anti-mouse IgG (diluted 1:10 in UW solution) for 30 min and were then washed twice with UW solution. Then the specimens were treated with 2.5% glutaraldehyde containing sodium cacodylate buffer (pH 7.4, 0.1 M), rinsed several times with the same buffer, and treated with 1% osmium tetroxide in the same buffer. They were dehydrated in graded ethanol, immersed in isoamylacetate, critical point-dried with CO₂, and coated with carbon using a CC-40F carbon coater (Meiwa Shouji, Tokyo, Japan). The cells were observed in FESEM by high-resolution backscatter electron (BSE) imaging using a YAG detector as well as secondary electron (SE) imaging. To quantify the number of gold particles, we counted them on five cells in randomly chosen 15 squares (2.25 cm²) using 8,000-fold magnified photographs.

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Distribution of GPI-80 on the surfaces of fMLP-stimulated neutrophils incubated in the culture plates
Apical surfaces
Very early after fMLP stimulation (1 min), which induces neutrophil activation, cells showing a spherical shape bore small numbers of short protrusions. At low magnification, GPI-80 was diffusely distributed on the surfaces of the cells (Fig. 1A ). With further incubation (15 min), many long protrusions appeared on the apical surfaces with increased spreading. GPI-80 was mainly detected on the peripheral surfaces of spreading cells (Fig. 1B) . The amount of GPI-80 on apical surfaces at 1 and 15 min did not differ significantly. At approximately 90 min of incubation, cells changed to a round shape with decreasing numbers of protrusions and GPI-80 was again distributed diffusely (Fig. 1C) . When we counted the number of gold particles as described in Materials and Methods, at 90 min of incubation GPI-80 was significantly reduced (140 ± 9.0/2.25 cm² in 8,000-fold magnification) compared to the very early stage of fMLP stimulation (412 ± 11.4/2.25-cm² in 8,000-fold magnification).

When the distribution of GPI-80 on the surfaces was determined more precisely with high magnification, in some parts of the specimen (Fig. 1E) GPI-80 was distributed diffusely, but in other parts in a clustered form (Fig. 1G and 1I) .

Ventral surfaces
We examined the distribution of GPI-80 on the ventral surfaces of cells adhering to the lower membranes after stripping off the upper membranes as described in Materials and Methods. Many flattened large projections were observed in some parts of the specimens, suggesting that these areas are the ventral surfaces that adhered to the lower membranes (Fig. 2A ). On the adhering surfaces very few GPI-80 were detected (Fig. 2B and 2C) . The reason for the scanty number of GPI-80 on these surfaces could not be ascribed to being ripped off when the two membranes were separated because no GPI-80 was detected on the surfaces of the lower membranes after being stripped from the upper membranes, and numerous CD18 were detected on the ventral surfaces of adhering cells (data not shown). On the other hand, GPI-80 in relatively small number was diffusely detected on the surfaces of small round projections, which may not have adhered to the lower membranes (Fig. 2D and 2E) .

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Figure 2. Localization of GPI-80 on the ventral cell surface. A typical ventral surface is shown in A. (B and D) higher magnifications of framed areas in A. (B and C) few GPI-80 were detected on the flattened surfaces of large projections, suggesting a tight cell-substratum contact area of the neutrophil (arrowheads). (D and E) on the small round projections, relatively small numbers of GPI-80 were detected (arrowheads). (A, B, and D) SE images. (C and E) BSE images of B and D, respectively. Bars, 1.0 µm (A), and 0.2 µm (B–E).

Clustered distribution of GPI-80 on the forward surfaces of neutrophils transmigrating through a Boyden chamber
We next examined the distribution of GPI-80 on the surfaces of neutrophils that passed through holes of a Boyden chamber. Many GPI-80 were detected on the forward surfaces of the migrating portions of neutrophils, resulting in a clustered distribution of GPI-80 on the migrating portions under low magnification (Fig. 3A , D-G , and I-L ). On the other hand, few GPI-80 were detected on the surfaces of the adherent portions of neutrophils still remaining in the upper chambers (Fig. 3B and 3C) . At higher magnification, many clustered GPI-80 were detected on the forward surfaces of transmigrating neutrophils (Fig. 3E 3G 3J and 3L) .

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Figure 3. Clustered distribution of GPI-80 on the forward surfaces of migrating neutrophils. (A–G) features of a migrating neutrophil observed from the apical sides of the upper chamber. (A) a complete picture of a migrating neutrophil. (B and D) higher magnifications of framed areas in A. Few GPI-80 are observed on the surface of the adherent portions in B and C. (E) BSE imaging of D. Clusters of GPI-80 are observable (arrowheads). (F) higher magnifications of framed areas in D. (G) BSE imaging of F. Clusters of GPI-80 are observed on the forward surfaces of a migrating neutrophil (arrowheads). (H–L) features of a migrating neutrophil observed from the ventral sides of the lower chamber. (H) a tip of a migrating neutrophil. (I and K) higher magnifications of framed areas in H and I, respectively. J and L are BSE imaging of I and K, respectively. Clusters of GPI-80 are observed on the forward surfaces of a migrating neutrophil (arrowheads). A, B, D, F, H, I, and K are SE images. C, E, G, J, and L are BSE images of B, D, F, I, and K, respectively. Bars, 10 µm (A), 0.3 µm (B, C, D, E, H, I, and J), and 0.1 µm (F, G, K, and L).

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study we have demonstrated the expression of a novel GPI-anchored protein on the surfaces of human neutrophils, tentatively designated GPI-80, that may regulate ß₂ integrin-mediated adhesion and transendothelial migration of human leukocytes [² ]. The most characteristic feature of our findings is the clustered expression of this GPI-anchored protein on the forward surfaces of migrating neutrophils (Fig. 3) . Because the number of gold particles on the surfaces of the adherent portions of neutrophils that remained in the upper chamber was reduced compared with that observed on the apical surfaces of fMLP-stimulated adhering neutrophils after 15 min of incubation (Fig. 1B) , GPI-80 may form clusters on the surfaces of migrating neutrophils, although it is still not clear whether or not the total amount of GPI-80 increased during migration. This result indirectly suggests that this GPI-anchored protein is involved in migration of neutrophils, although its actual role in leukocyte migration is still unknown. In our literature survey, we found no electron microscope studies showing that cell adhesion molecules or their regulating molecules, such as integrins or the urokinase-type plasminogen activator receptor (uPAR), that may be involved in leukocyte migration, are localized on the surfaces of migrating leukocytes in a manner such as shown in the present study. Therefore, at present we are unable to discuss our result that the GPI-80 is clustered on the forward surfaces of migrating neutrophils in terms of the relationships between GPI-80 and integrins or the uPAR. However, it has been well documented that changes in expression or new epitope appearance of integrins on leukocytes occurs depending on their activation state [^{11 12 13} ]. Furthermore, localization of GPI-anchored proteins such as uPAR and lipopolysaccharide (LPS)/LPS binding protein receptor (CD14) on leukocyte surfaces that may be involved in regulation of leukocyte migration changes with activation of the cells [¹⁴ ]. With this in mind, it seems highly possible that molecules involved in leukocyte migration may cluster on the forward surfaces of migrating leukocytes as shown in the present study of GPI-80, or they disappear from these areas. Further immuno-electron microscopy studies on the localization of other molecules involved in leukocyte migration may clarify the significance of our results on the characteristic localization of GPI-80.

Sparse distribution of GPI-80 on ventral surfaces adhering to ligands on the membranes was an unexpected result, since in a previous study we demonstrated that 3H9 (a mAb to GPI-80) modulates ligand binding avidity of ß₂ integrin, which should be abundantly localized on the surfaces of leukocytes adhering to ligands. Sparse distribution of GPI-80 on the ventral adhering surfaces and clustering one on the forward surfaces of migrating neutrophils may suggest that GPI-80 is not actually involved in modulation of cell adherence but rather only of cell locomotion. However, we cannot be sure of this, because the experimental procedure we used to observe GPI-80 expression on ventral surfaces in the present study is not the ideal condition for this purpose. As described in Materials and Methods, the upper membranes on which neutrophils were placed were 10 µm thick. Under this condition, we actually investigated the ventral surfaces of neutrophils that migrated through the hole of the upper membranes in distances of at least 10 µm, but not those of neutrophils adhering to the surface of the upper membranes. To this end, we will need revised techniques.

In conclusion, the present result showing a clustered distribution of GPI-80 on the forward surfaces of migrating neutrophils may suggest not only a role for this novel GPI-anchored protein in leukocyte migration but also a certain role for GPI-anchored proteins in general. Molecules in this group are easily able to form clusters because of their high lateral mobility when such changes are required for certain biological activities of cell membranes.

Received August 10, 1999; revised June 22, 2000; accepted June 23, 2000.

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