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(Journal of Leukocyte Biology. 2002;71:205-211.)
© 2002 by Society for Leukocyte Biology

Cross-linking of GPI-80, a possible regulatory molecule of cell adhesion, induces up-regulation of CD11b/CD18 expression on neutrophil surfaces and shedding of L-selectin

Hiroshi Yoshitake, Yuji Takeda, Takeaki Nitto and Fujiro Sendo

Department of Immunology and Parasitology, 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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ABSTRACT
 
Previously, we described a novel glycosylphosphatidyl inositol (GPI)-anchored glycoprotein (designated GPI-80) on human neutrophils and monocytes that may regulate ß2 integrin-dependent neutrophil adherence and migration. However, the mechanism regulating ß2 integrin remains to be clarified. To study this, we examined changes in ß2 integrin expression and function caused by cross-linking GPI-80. GPI-80 cross-linking induced up-regulation of CD11b/CD18 (Mac-1) expression on neutrophil surfaces and shedding of L-selectin, which depends on tyrosine phosphorylation and cytoskeleton remodeling. Furthermore, the cross-linking enhanced fMLP-induced human neutrophil adherence. These results suggest that GPI-80 may be a regulator of ß2 integrin in neutrophils.

Key Words: integrin • adherence • uPAR • DAF


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INTRODUCTION
 
Neutrophils play important roles in inflammation and the immune response, not only through their effector mechanisms using bioactive molecules but also through modulation of inflammation and the immune response by cytokines that they produce. For these activities, neutrophil extravasation is essential. In this process, neutrophils first bind loosely to endothelial cells through interaction between L-selectin on the neutrophils or P-selectin on the endothelial cells and their ligands, and then the neutrophils roll on the endothelium in the direction of the bloodstream. Next, the neutrophils attach firmly to the endothelial cells through interaction between ß2 integrins on the neutrophils and their ligands, and then the cells pass through the basal membrane and reach the extravascular tissues. This outline of neutrophil extravasation has been documented in the last 10 years [1 2 ]. However, the precise molecular mechanisms involved in sequential adhesion and deadhesion of neutrophils to the endothelium have not yet been clarified.

During our study about the mechanisms of neutrophil extravasation, we established a monoclonal antibody (mAb), 3H9, that modulates ß2 integrin-dependent neutrophil adhesion [3 ]. We cloned the molecule recognized by 3H9, identified it as a novel 80-kD glycosylphosphatidyl inositol (GPI)-anchored protein, and designated it as GPI-80 [4 ].

It is well known that GPI-anchored proteins transduce biochemical signals into the cytoplasm, but the mechanisms are still obscure because GPI-anchored proteins do not have intracellular domains. Conversely, clustering of GPI-anchored protein on cell surfaces induces intracellular signaling [5 6 7 ]. Previously, we demonstrated that cross-linking GPI-80 on neutrophil surfaces by the mAb 3H9 induces intracellular tyrosine phosphorylation [8 ].

Similar to GPI-80, GPI-anchored proteins such as urokinase-type plasminogen-activator receptor (uPAR, CD87), Fc{gamma}RIIIb (CD16b), and CD14, regulate the functions of ß2 integrins on neutrophil surfaces [9 ]. Cross-linking neutrophil surface uPAR or Fc{gamma}RIIIb by their antibodies induces an increase in the expression of CD11b on the surfaces of the cells [10 , 11 ]. In this study, we examined whether cross-linking GPI-80, which possibly regulates ß2 integrin-dependent neutrophil adhesion, modulates the expression of Mac-1 (CD11b/CD18) or L-selectin on neutrophil surfaces as does cross-linking other modulators of neutrophil function, uPAR, or Fc{gamma}RIIIb.


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MATERIALS AND METHODS
 
Reagents
Used reagents were purchased from the following companies: Dextran 200,000 and paraformaldehyde, Wako Pure Chemical Industries, Osaka, Japan; Ficoll-Paque, Pharmacia Biotech, Uppsala, Sweden; Hanks’ balanced saline solution (HBSS), Nissui Pharmaceutical Co., Tokyo, Japan; and N-formyl-methionyl-leucyl-phenylalanine (fMLP), genistein, and cytochalasin B, Sigma Chemical Co., St. Louis, MO.

Antibodies
The anti-GPI-80 mAb 3H9 [mouse immunoglobulin G (IgG1)] was established and characterized as described previously [3 ]. The F(ab')2 fragment of 3H9 was prepared by the methods of Lamoyi and Nisonoff [12 ]. Fluorescein isothiocyanate (FITC)-conjugated 3H9 F(ab')2 was prepared by a modified method described by Johnson et al. [13 ]. mAb TCY-3 (mouse IgG1), specific for Trypanosoma cruzi, was used for control studies [14 ]. CBRM1/5 mAb (mouse IgG1, specific for a subpopulation of neutrophils CD11b) was a gift from Dr. Timothy A. Springer (Harvard Medical School, Boston, MA) [15 ]. Antibodies were purchased from the following companies: FITC-conjugated anti-CD11b mAb and FITC-conjugated goat anti-mouse IgG Fc region-specific antibody F(ab')2 fragment, Immunotech A Coulter Co., Marseille, France; FITC-conjugated anti-CD55 mAb and r-phycoerythrin (RPE)-conjugated anti-CD18 mAb, PharMingen, San Diego, CA; RPE-conjugated anti-CD11b mAb and RPE-conjugated anti-HLA class I mAb, DAKO, Glostrup, Denmark; RPE-conjugated anti-Leu-8 (L-selectin) mAb, Becton Dickinson Immunocytometry Systems, San Jose, CA; and rabbit anti-mouse IgG (H+L) antibody F(ab')2 fragment and negative control mouse IgG F(ab')2 fragment, Jackson ImmunoResearch Laboratories, West Grove, PA.

Preparation of human neutrophils
Heparinized venous blood from healthy volunteers was sedimented through Dextran 200,000. The leukocyte-rich supernatant (buffy coat) was centrifuged at 450 x g for 5 min and washed with phosphate-buffered saline (PBS). Neutrophils were isolated from the buffy coat using Ficoll-Paque as described previously [16 ], and residual erythrocytes were lysed by hypotonic shock. The isolated neutrophils were resuspended in HBSS. The isolated neutrophils were >95% pure.

Cross-linking GPI-80 or decay accelerating factor (DAF) by their antibodies
The isolated neutrophils were treated for 30 min at 4°C with 10 µg/ml 3H9 F(ab')2 or a 1/30 dilution of anti-CD55 (DAF) mAb in HBSS and then washed twice with cold HBSS. Next, the cells were treated for 30 min at 4°C with 20 µg/ml rabbit anti-mouse IgG F(ab')2 in HBSS and then at 37°C for various times. After incubation, the cells were fixed for 30 min at 4°C with 2% paraformaldehyde in PBS and then washed twice with PBS.

Labeling cells
The fixed cells were treated for 30 min at 4°C with FITC or RPE-conjugated anti-CD11b, RPE-conjugated anti-CD18, RPE-conjugated Leu-8, or HLA class I antibody in PBS, after which the cells were washed twice with PBS. The CBRM1/5 mAb, bound to a subpopulation of CD11b, was labeled as follows. The fixed cells were treated with 30 µg/ml control mouse IgG F(ab')2 fragment for 30 min at 4°C in PBS to block remaining binding sites of rabbit anti-mouse IgG F(ab')2 used for cross-linking. After washing twice with PBS, the cells were incubated for 30 min at 4°C in PBS containing 3 µg/ml CBRM1/5 mAb and 1.5 µg/ml FITC-conjugated goat anti-mouse IgG Fc region-specific Ab F(ab')2 fragment in PBS and then washed twice with PBS.

Flow cytometry
The expression of CD11b, CD18, L-selectin, and HLA class I on the surfaces of the cells was measured with a FACSCalibur (Becton Dickinson Immunocytometry Systems). Dead cells and debris were excluded from the analysis by forward- and side-scatter gating.

Confocal laser microscopy
Isolated neutrophils were treated for 30 min at 4°C with FITC-conjugated 3H9 F(ab')2 fragment or FITC-conjugated anti-CD55 mAb in HBSS and then washed twice with cold HBSS. The cells were treated for 30 min at 4°C with 20 µg/ml rabbit anti-mouse IgG F(ab')2 in HBSS and then incubated for 30 min at 37°C or 4°C. After incubation, the cells were fixed for 30 min at 4°C with 2% paraformaldehyde in PBS and then washed with PBS. The fixed cells were seeded on Vectabond (Vector Lab, Burlingame, CA)-coated coverslips. The specimens were mounted in PBS and examined with a confocal laser microscope (TCS, Lica, Hiderberg, Germany).

Neutrophil adherence assay
Details of this assay have been described elsewhere [16 ]. Briefly, Falcon 3072 plates (Becton Dickinson Labware, Franklin Lakes, NJ) were coated for 2 h at 37°C with 50 µg/ml fibrinogen and then washed with PBS. Neutrophil suspensions in 200 µl (5x105 cells/well), which had been treated with antibodies to GPI-80, DAF, or TCY-3 and then with anti-mouse IgG mAbs or stimulated with 1 x 10-7 M fMLP suspensions (5x105 cells/well) in HBSS, were added to fibrinogen-coated plates and incubated at 37°C for various times. The percentage of adherence was calculated using the following fomula: {% adhesion=[(OD570 nm after GPI-80 or DAF cross-linking, or fMLP stimulation-OD570 nm after TCY-3 cross-linking)/OD570 nm after TCY-3 cross-linking]x100}.

Statistical analysis
Analysis of the statistical significance of differences between two mean values was performed using the Student’s t-tests. P values < 0.05 were considered significant.


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RESULTS
 
Enhancement of CD11b expression and down-modulation of L-selectin on neutrophil surfaces by cross-linking GPI-80 with mAb 3H9
We examined the change in CD11b expression on neutrophil surfaces after cross-linking GPI-80 with 3H9 and anti-mouse IgG antibody. As shown in Figure 1 A , cross-linking GPI-80 enhanced the cell-surface expression of CD11b in a time-dependent manner for up to 5 min of incubation when it reached a plateau. As shown by Borregaard et al. [17 ], neutrophil surface expression of CD11b is up-regulated by fMLP treatment, and we found a similar pattern with GPI-80 cross-linking. Neither treatment with 3H9 alone in the absence of cross-linking with anti-mouse IgG antibody nor treatment with anti-mouse IgG antibody alone changed the expression of CD11b. In addition, cross-linking GPI-80 did not change the expression of HLA on neutrophil surfaces.



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  		Figure 1. Up-regulation of CD11b expression on neutrophil surfaces and shedding of L-selectin by cross-linking GPI-80. After cross-linking GPI-80, the cells were incubated for 0, 1, 3, 5, 15, or 30 min at 37°C. Then the neutrophils were labeled with FITC-conjugated anti-CD11b mAb, RPE-conjugated anti-L-selectin mAb, or anti-HLA mAb. (A) Changes in CD11b expression. (•) CD11b expression after GPI-80 cross-linking [CD11b XL(+)]. ({circ}) CD11b expression after treatment with 3H9 alone [CD11b XL(-)]. ({blacklozenge}) CD11b expression after fMLP stimulation [CD11b fMLP(+)]. ({triangleup}) HLA expression after GPI-80 cross-linking [HLA XL(+)]. ({square}) CD11b expression after treatment with anti-mouse IgG Ab alone (CD11b a-mIg). (B) Changes in L-selectin expression. (•) L-selectin expression after GPI-80 cross-linking [L-s XL(+)]. ({circ}) L-selectin expression after treatment with 3H9 alone [L-s XL(-)]. ({blacklozenge}) L-selectin expression after fMLP treatment [L-s fMLP(+)]. ({triangleup}) HLA expression after GPI-80 cross-linking [HLA XL(+)]. The data shown are the increases in mean fluorescence intensity (MFI; i.e., MFI after incubation/MFI before incubation) ± SE (A, n=5; B, n=3). **, P < 0.01; *, P < 0.05 compared with control without GPI-80 cross-linking.

CD11b forms a heterodimer with CD18 on leukocyte surfaces [18 ]. Therefore, we then examined the expression of CD18 after GPI-80 cross-linking and found that it was enhanced like that of CD11b (unpublished results).

Conversely, GPI-80 cross-linking decreased the expression of L-selectin on neutrophil surfaces in a time-dependent manner for up to 5 min, at which time it reached a plateau. As demonstrated by Borregaard et al. [17 ], treatment with fMLP decreases L-selectin expression, and we found a similar pattern with GPI-80 cross-linking. L-selectin expression did not change significantly by treatment with 3H9 alone (Fig. 1B) .

We examined the effect of 3H9 concentration on the increase in CD11b expression by GPI-80 cross-linking and found that it increased in a concentration-dependent manner up to 10 µg/ml 3H9 when it reached a plateau (Fig. 2 ).



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  		Figure 2. Effect of 3H9 concentration on CD11b expression induced by cross-linking GPI-80. After cross-linking GPI-80 with 0, 1, 2, 3, 5, 7, 10, 15, or 20 µg/ml 3H9 F(ab')2 fragments and rabbit anti-mouse IgG F(ab')2 (20 µg/ml), the cells were incubated for 15 min at 37°C. (•) CD11b expression after GPI-80 cross-linking [XL(+)]. ({circ}) CD11b expression after treatment with 3H9 alone [XL(-)]. The data shown are the increases in MFI (i.e., MFI in the presence of 3H9/MFI in the absence of 3H9) ± SE (n=3). *, P < 0.05 between cross-linking and no cross-linking.

Change in localization of GPI-80 on neutrophil surfaces after cross-linking with 3H9
Using confocal laser microscopy, we examined the localization of GPI-80 after being cross-linked. When neutrophils were treated with 3H9 alone, GPI-80 was broadly spread on entire neutrophil surfaces, before incubation and after incubation for 30 min at 37°C (Fig. 3A and B). Conversely, after GPI-80 was cross-linked by the addition of antibodies to mouse Ig, it formed a patchy pattern before incubation (Fig. 3C) and a cap after incubation for 30 min at 37°C (Fig. 3D) , similar to uPAR after being cross-linked [19 ].



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  		Figure 3. Change in localization of GPI-80 on the surface of neutrophils after cross-linking GPI-80 by 3H9. GPI-80 on the cells was labeled with FITC-conjugated 3H9 and observed by confocal laser microscopy. (A) GPI-80 was not cross-linked, and the cells were not incubated. (B) GPI-80 was not cross-linked, and the cells were incubated for 30 min at 37°C. (C) GPI-80 was cross-linked, and the cells were not incubated. (D) GPI-80 was cross-linked, and the cells were incubated for 30 min at 37°C.

The effect on cell-surface expression of CD11b by cross-linking another GPI-anchored protein, DAF (CD55)
We determined whether cross-linking another cell-surface GPI-anchored protein, DAF, affects cell-surface expression of CD11b to ascertain if the observed phenomena may be ascribed to cross-linking GPI-anchored proteins in general. Before doing this, we examined the cell-surface localization of DAF after cross-linking it and incubating at 37°C. Before cross-linking DAF, it was observed evenly on the entire cell surface (Fig. 4 A ).



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  		Figure 4. Localization of DAF on neutrophil surfaces after cross-linking with a relevant mAb. (A) No cross-linking. (B) Cross-linking without incubation. (C) Cross-linking and incubation for 30 min at 37°C.

We examined the change in the localization of DAF on cell surfaces caused by its cross-linking. As shown in Figure 4C , the result was the same as that with GPI-80 in that cross-linking DAF followed by incubation for 30 min at 37°C resulted in cap formation. These results show that cross-linking GPI-80 and DAF induces similar localization of the proteins, prompting us to examine the effect of DAF cross-linking on the expression of CD11b on neutrophil surfaces. Although, as already shown, cross-linking GPI-80 increased the cell-surface expression of CD11b, cross-linking DAF had no effect on CD11b expression (Fig. 5 ), suggesting that cross-linking GPI-anchored proteins in general does not induce enhanced expression of CD11b. To eliminate the possibility that the results we obtained might be ascribed to a difference in the amounts of CD55 and GPI-80 on neutrophil surfaces, we compared them by flow cytometry and found that there was not a large difference in their amounts on neutrophil surfaces (unpublished results).



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  		Figure 5. Effect of DAF cross-linking on CD11b expression on neutrophil surfaces. After GPI-80 or DAF was cross-linked, the cells were incubated for 0, 1, 3, 5, 15, or 30 min at 37°C. The cells were fixed and labeled with RPE-conjugated anti-CD11b mAb. (•) CD11b expression after DAF cross-linking [DAF XL(+)]. ({circ}) CD11b expression after treatment with anti-DAF mAb alone [DAF XL(-)]. ({blacksquare}) CD11b expression after GPI-80 cross-linking [GPI-80 XL(+)]. ({square}) CD11b expression after treatment with 3H9 alone [GPI-80 XL(-)] . ({blacktriangleup}) CD11b expression after TCY-3 cross-linking [TCY-3 XL(+)]. ({blacklozenge}) CD11b expression after fMLP stimulation [fMLP(+)]. The data shown are the changes in MFI (i.e., MFI after incubation/MFI before incubation) ± SE (n=4). *, P < 0.05 compared with no DAF cross-linking.

Effect of a tyrosine-kinase inhibitor, genistein, and an inhibitor of actin polymerization, cytochalasin B, on the enhanced expression of CD11b caused by cross-linking GPI-80
Previously, we showed that cross-linking GPI-80 induces tyrosine phosphorylation of a 34-kD protein that is inhibited by cytochalasin B [8 ]. Therefore, we examined the effects of a tyrosine phosphorylation inhibitor, genistein, and an inhibitor of actin polymerization, cytochalasin B, on the enhanced expression of CD11b on cell surfaces caused by GPI-80 cross-linking. As shown in Figure 6 , both agents inhibited the enhanced expression of CD11b in a concentration-dependent manner, suggesting that tyrosine phosphorylation and cytoskeleton remodeling are involved in the enhancement of CD11b expression on neutrophil surfaces caused by GPI-80 cross-linking.



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  		Figure 6. Effects of genistein and cytochalasin B on the up-regulation of CD11b expression induced by GPI-80 cross-linking. After treating with 3H9 F(ab')2 fragments, the cells were treated with 50 or 100 µg/ml genistein or 5 or 10 µg/ml cytochalasin B for 15 min at room temperature. After GPI-80 cross-linked, the cells were incubated for 15 min at 37°C in HBSS containing genistein or cytochalasin B at the indicated concentrations. The data are shown as %MFI [i.e., (MFI with GPI-80 cross-linking in the presence of inhibitor-MFI without GPI-80 cross-linking in the presence of inhibitor)/(MFI with GPI-80 cross-linking in the absence of inhibitor-MFI without GPI-80 cross-linking in the absence of inhibitor)] x 100 ± SE (A, n=4; B, n=3). **, P < 0.01; *, P < 0.05 compared with no inhibitors.

Effect of GPI-80 cross-linking on ß2 integrin-dependent neutrophil adherence
It is well documented that enhanced expression of ß2 integrins on cell surfaces is not necessarily followed by increased cell adhesion [20 , 21 ] and that activation epitopes of ß2 integrins are critically important for ß2 integrin-dependent cell adhesion [15 ]. Thus, we examined whether activation epitopes of CD11b were increased by GPI-80 cross-linking. For this, we used CBRM1/5 mAb [15 ]. Figure 7 shows that this cross-linking up-regulated the expression of CD11b recognized by CBRM1/5 on neutrophil surfaces, although the rate of up-regulation was less than that induced by fMLP treatment.



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  		Figure 7. Up-regulation of CBRM1/5-recognized CD11b expression on the neutrophil surfaces by cross-linking GPI-80. After cross-linking GPI-80, the cells were incubated for 0, 1, 3, 5, 15, or 30 min at 37°C. Then the cells were labeled with CBRM1/5 or TCY-3 and FITC-conjugated anti-mouse IgG Fc region-specific Ab. (•) CBRM1/5 expression after GPI-80 cross-linking [XL(+)]. ({circ}) CBRM1/5 expression after treatment with 3H9 alone [XL(-)]. ({triangleup}) TCY-3 expression after GPI-80 cross-linking [XL(+) TCY-3]. ({blacksquare}) CBRM1/5 expression after fMLP stimulation [fMLP(+)]. The data shown are the increases in MFI (i.e., MFI after incubation/MFI before incubation) ± SE (n=4). ***, P < 0.005; **, P < 0.01; *, P < 0.05 compared with no GPI-80 cross-linking.

Next, we determined whether GPI-80 cross-linking augments ß2 integrin-dependent neutrophil adherence. As shown in Figure 8 , fMLP treatment enhanced neutrophil adherence on a plate precoated with fibrinogen, but GPI-80 cross-linking had no effect, suggesting that GPI-80 cross-linking itself does not affect ß2 integrin-dependent adherence, although it enhances the expression of the ß2 integrin-activation epitope.



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  		Figure 8. Effect of GPI-80 cross-linking on ß2 integrin-dependent neutrophil adherence. (•) GPI-80 cross-linking [GPI-80 XL(+)]. ({circ}) Treatment with 3H9 alone [GPI-80 XL(-)]. ({triangleup}) DAF cross-linking [DAF XL(+)]. ({blacksquare}) fMLP stimulation [fMLP(+)]. The data shown are % adherence {i.e., [OD values after GPI-80 cross-linking (+) or (-), DAF cross-linking (+), or fMLP stimulation-OD values of TCY-3 cross-linking (+)]/OD values of TCY-3 cross-linking (+)} x 100 ± SE (n=3). *, P < 0.02 compared with no GPI-80 cross-linking.

Although it was shown that GPI-80 cross-linking itself does not affect ß2 integrin-dependent adherence (Fig. 8) , we examined the priming effects of GPI-80 cross-linking on fMLP-induced human neutrophil adherence. Figure 9 shows that cross-linking GPI-80 augmented fMLP-induced neutrophil adherence at 30 min of incubation, whereas cross-linking another GPI-anchored protein on human neutrophils, DAF, did not affect adherence.



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  		Figure 9. Enhancement of fMLP-induced neutrophil adherence by GPI-80 cross-linking. After cross-linking GPI-80 or DAF, neutrophil suspensions and 200 µl HBSS containing 1 x 10-9 M fMLP were added to fibrinogen-coated plates, which were incubated at 37°C for 15, 30, 60, and 120 min. (•) GPI-80 cross-linking. ({square}) DAF cross-linking. The data shown are % adherence [i.e., (OD values after GPI-80 or DAF cross-linking-OD values of TCY-3 cross-linking)/OD values of TCY-3 cross-linking] x 100 ± SE (n=3). *, P < 0.05 compared with TCY-3 cross-linking.


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DISCUSSION
 
To elucidate the mechanisms by which a novel GPI-anchored protein on neutrophils, GPI-80, regulates ß2 integrin-dependent neutrophil adherence, we examined the effect of cross-linking GPI-80 on the cell-surface expression of Mac-1 by human neutrophils. We found that cross-linking GPI-80 enhances the expression of Mac-1 and down-regulates that of L-selectin on neutrophils. These results strengthen the possibility that GPI-80 regulates ß2 integrin-mediated cell adherence. Recently, we have demonstrated that GPI-80 forms clusters on the forward surfaces of transmigrating neutrophils [22 ] and now show that cross-linking GPI-80 induces its cluster formation (Fig. 3) . Some other GPI-anchored proteins, such as uPAR (CD87), Fc{gamma}RIIIb (CD16b), and CD14, modulate adherence and migration of neutrophils [9 ], and cross-linking uPAR or Fc{gamma}RIIIb with relevant antibodies enhances Mac-1 expression on neutrophil surfaces [10 , 11 ]. The present results, cross-linking GPI-80, mimic those obtained with these other GPI-anchored proteins, which regulate adherence. Although we have not yet identified the GPI-80 ligand, cross-linking GPI-80, which results in its clustering (Fig. 3) , may mimic the physiological effect of the ligand binding to GPI-80, which likely causes its clustering.

Conversely, with respect to the modulation of L-selectin shedding by cross-linking GPI-anchored proteins that regulate adhesion, varying results have been obtained. In this study and in the case of cross-linking certain alleles (Fc{gamma}RIIaR131) of Fc{gamma}RIIa [11 ], cell-surface expression of L-selectin was down-regulated. However, cross-linking another allele (Fc{gamma}RIIaH131) of Fc{gamma}RIIa, Fc{gamma}RIIIb [11 ], or uPAR did not induce down-regulation of L-selectin, although in the uPAR cross-linking experiment, expression of L-selectin was down-regulated even by treatment with a control antibody [10 ], which was not observed in the other cross-linking studies including the one described here. Although the reason for these conflicting results remains to be clarified, each GPI-anchored protein that regulates cell adhesion may work at different stages of leukocyte extravasation.

Previously, it was shown that fMLP-induced degranulation of primary and secondary granules is inhibited by genistein [23 ]. As shown in Figure 6A , genistein inhibits the up-regulation of CD11b expression on neutrophil surfaces caused by GPI-80 cross-linking, suggesting that tyrosine phosphorylation is required for degranulation following GPI-80 cross-linking. Conversely, in contrast to genistein, cytochalasin B enhances fMLP-induced degranulation of primary and secondary granules [23 ]. However, as shown in Figure 6B , degranulation caused by cross-linking GPI-80 was inhibited by cytochalasin B. It has been shown that cytoskeletal reorganization is required for capping GPI-anchored proteins in lymphocytes [24 ]. Under these situations, cytochalasin B may affect capping formation of GPI-80 that may be required for signal transduction for Mac-1 expression enhancement.

Although not only was total expression of Mac-1 enhanced (Fig. 1) , but expression of an activation epitope of CD11b (CBRM1/5) was also increased by GPI-80 cross-linking (Fig. 7) , and ß2 integrin-dependent neutrophil adherence was not affected (Fig. 8) . This suggests that cross-linking GPI-80 does not induce full activation of neutrophils that are required for ß2 integrin-dependent adherence of these cells and that expression of an activation epitope recognized by CBRM1/5 is a required but not sufficient condition for ß2 integrin-dependent cell adhesion. The finding that cross-linking GPI-80 primes neutrophils for fMLP-induced adherence (Fig. 9) but does not itself induce adherence (Fig. 8) supports this notion. Our preliminary results showing that cross-linking GPI-80 did not change the topographical localization of CD18 on neutrophil surfaces (unpublished results) may further explain the condition of neutrophils whose GPI-80 is cross-linked. We should note here that as shown in our previous study, treatment of neutrophils with the mAb to GPI-80, 3H9, modulates the fMLP-induced adherence of neutrophils [3 ], but 3H9 in the absence of additional neutrophil stimulation does not induce adherence, indicating that GPI-80 is a regulator of cell adhesion. Furthermore, in the present study, cross-linking GPI-80 augmented fMLP-induced neutrophil adherence (Fig. 9) , although by itself it did not induce adherence (Fig. 8) . This suggests that cross-linking GPI-80 has a priming activity on neutrophil function(s).

GPI-anchored proteins on lymphocytes are localized in a detergent-insoluble cell-membrane fraction [25 ], which is called a "microdomain" or "raft" containing an abundance of the signaling molecules [26 , 27 ]. Rafts are considered to be involved in signal transduction into the cytoplasm through cross-linking GPI-anchored proteins, although the precise mechanisms remain to be clarified [28 ]. By cross-linking with relevant antibodies, GPI-anchored proteins become focused on one point of the cell-surface membrane, forming a capping. Rafts are translocated to this point, and signaling proteins in rafts are phosphorylated [29 ]. Although it is not known whether rafts exist on neutrophil-surface membranes, cross-linking GPI-80 and DAF on human neutrophils by relevant antibodies causes them to move to the same point of cell-surface membranes and form a cap (Figs. 3 and 4) . However, DAF cross-linking did not induce an increase in cell-surface expression of Mac-1 (Fig. 5) in contrast to the effect of GPI-80 cross-linking (Fig. 1A) , suggesting that the phosphorylation induced by cross-linking GPI-anchored proteins cannot be explained by the raft phenomena, but certain mechanisms by which specificity of each GPI-anchored protein are determined.

In summary, the present result that cross-linking GPI-80 enhances Mac-1 expression on neutrophil surfaces and the adherence suggests that this GPI-anchored protein is involved in the regulation of ß2 integrin-dependent neutrophil adherence and consequently its transendothelial migration.


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ACKNOWLEDGEMENTS
 
We thank Dr. Timothy A. Springer, Harvard Medical School, for the gift of the monoclonal antibody, CBRM1/5.

Received June 14, 2001; revised September 18, 2001; accepted September 19, 2001.


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K.-S. Choi, J. Garyu, J. Park, and J. S. Dumler
Diminished Adhesion of Anaplasma phagocytophilum-Infected Neutrophils to Endothelial Cells Is Associated with Reduced Expression of Leukocyte Surface Selectin
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