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A peptide derived from the intercellular adhesion molecule-2 regulates the avidity of the leukocyte integrins CD11b/CD18 and CD11c/CD18.

beta 2 integrin (CD11a,b,c/CD18)-mediated cell adhesion is required for many leukocyte functions. Under normal circumstances, the integrins are nonadhesive, and become adhesive for their cell surface ligands, the intercellular adhesion molecules (ICAMs), or soluble ligands such as fibrinogen and iC3b, when leukocytes are activated. Recently, we defined a peptide derived from ICAM-2, which specifically binds to purified CD11a/CD18. Furthermore, this peptide strongly induces T cell aggregation mainly mediated by CD11a/CD18-ICAM-1 interaction, and natural killer cell cytotoxicity. In the present study, we show that the same ICAM-2 peptide also avidly binds to purified CD11b/CD18, but not to CD11c/CD18. This binding can be blocked by the CD11b antibody OKM10. The peptide strongly stimulates CD11b/CD18-ICAM-1-mediated cell aggregations of the monocytic cell lines THP-1 and U937. The aggregations are energy and divalent cation-dependent. The ICAM-2 peptide also induces CD11b/CD18 and CD11c/CD18-mediated binding of THP- 1 cells to fibrinogen and iC3b coated on plastic. These findings indicate that in addition to induction of CD11a/CD18-mediated cell adhesion, the ICAM-2 peptide may also serve as a "trigger" for high avidity ligand binding of other beta 2 integrins.

The leukocyte integrins need to be activated in order to become adhesive. The molecular mechanisms involved in the activation of CDll/CD18 are incompletely understood. Quantitative as well as qualitative changes occur in these receptors after cell activation. Phorbol ester treatment (Patarroyo et al., 1985;, or crosslinking the T cell receptor on lymphocytes ) induces a high avidity state of CD1 la/CD18 for ICAM-1, without any substantial increase in lymphocyte surface expression of this integrin. CD1 la/CD18 can also be activated by monoclonal antibodies reacting with a variety of leukocyte cell surface glycoproteins (van Kooyk et al., 1989;Koopman et al., 1990;Kansas and Tedder, 1991), evidently through "inside-out" signaling. In addition, certain monoclonal antibodies against the ct chain of CDlla/CD18 has been shown to be able to induce CDlla/CD18-dependent homotypic T cell adhesion (Koopman et al., 1992;Landis et al., 1993), indicating that integrin activation could also be induced by direct ligand binding from the outside of the cells.
Unlike CDlla/CD18, the amount of cell surface CDllb/ CD18 and CD1 lc/CD18 on granulocytes and monocytes can be rapidly upregulated by translocation of these two receptors from an intracellular pool to the cell surface in response to cell activation Bainton et al., 1987). However, the change in CD1 lb/CD18 surface expression that occurs after stimulation does not parallel the kinetics or magnitude of cell adhesion Lo et al., 1989). Thus, CD1 lb/CD18 is hypothesized to undergo additional qualitative conformational changes that facilitate adhesion Lo et al., 1989;Philips et al., 1988;Vedder and Harlan, 1988). The phorbol ester and adenine nucleotide ADP-induced functional modulation of CDllb/CD18 appears to involve allosteric or qualitative remodeling of the receptor characterized by the formation of activation-dependent neoantigenic epitopes Altieri and Edgington, 1988b). Alternatively, the enhanced avidity of CDllb/CD18 could be due to a signal to cluster the receptors in the plane of the membrane (Detmers et al., 1987).
Recent work has demonstrated that ICAM-1, ICAM-2, and ICAM-3 provide important costimulatory signals via their adhesive interactions with the CD11a/CD18 complex during the CD3/TCR-mediated activation of resting T cells (van Seventer et al., 1990;Damle et al., 1992;Hern/~ndez-Caselles et al., 1993). Furthermore, ICAM-1 has been shown to be able to induce the high affinity state of CD1 la/CD18 characterized by expression of the m_Ab 24 epitope (Cabanas and Hogg, 1993), which has been thought to be a "reporter" of the activated state of CDlla/CD18 (Dransfield et al., 1990). However, there is no direct evidence for inducing a high avidity state of leukocyte integrins by direct ligand binding.
ICAM-2 is the second ligand found for CDlla/CD18 (Staunton et al., 1989). The external portion of this molecule consists of two immunoglobulin domains, which have 34% identity in amino acid sequences with the two NH2terminal domains of ICAM-1 and ICAM-3. To identify the binding site(s) in ICAM-2 for CDlla/CD18, we recently synthesized several peptides from the first immunoglobulin domain of ICAM-2, and characterized a 22-amino acid long peptide, PI, which specifically binds to CDlla/CD18 (Li et al., 1993b). Furthermore, the peptide strongly stimulates blood T cell aggregation mainly mediated by CD1 ladCD18-ICAM-1 interaction, and natural killer cell cytotoxicity (Li et al., 1993a). In the present study, we report that the ICAM-2 peptide also binds to purified CD1 lb/CD18, but not to CDllc/ CD18. The peptide strongly induces CD1 lb/CD18-ICAM-1mediated homotypic cell adhesion of monocytic cell lines, and CDllb/CD18 and CDllc/CD18-mediated binding of leukocytes to fibrinogen and iC3b.

Peptide Synthesis
Peptides were synthesized on a model 430A peptide synthesizer, using Fmoc-chemistry (Applied Biosystems, Inc., Foster City, CA). The structures were confirmed by FAB-mass spectrometric analysis (JEOL SX-102). The sequence of the ICAM-2 peptide P1 is GSLEVNCSTTCNQPEVG-GLETS. The amino acids of the control peptide P8 are the same as those of P1, but in random order, except that the two conserved cysteine residues are in the right positions (Li et al., 1993b). After synthesis, the PI and P8 peptides were reduced with dithiothreitol (1:1.5 by wt/wt, in 0.1 M NaHCO3, pH 8.0) for 1.5 h at room temperature, and purified by high pressure liquid chromatography, using a reverse phase column (Waters Cis). A COOH-terminal tyrosine was added to the peptides to enable t25I-labeling (Li et al., 1993b).
lysates by affinity chromatography on monoclonal antibody LM2/1-Sepharose CL 4B and 3.9-Sepharose CL 4B, respectively, and eluted at pH 11.5 in the presence of 2 mM MgCI2 and 1% n-octyl glucoside . The purity of the proteins were checked by polyacrylamide gel electropboresis (PAGE) in the presence of SDS (Laemmli, 1970). The heterodimeric forms of the purified integrins were examined by running the preparations once again through the same affinity columns and checking the eluates by SDS-PAGE. The proteins were diluted 1:10 with 25 mM Tris, pH 8.0, 150 mM NaCI and 2 mM MgCI2, and attached to flat-bottomed, 96-well mierotiter plates (Dynatech Laboratories, VA) by overnight incubation at 4°C. The wells were blocked with 1% BSA for 1 h at room temperature. 12 pmol of 125I-labeled P1, labeled by using the chloramine-T method (Greenwood et al., 1963), was added to each well in 40 td of 0.15 M NaC1, 0.01 M sodium phosphate, pH 7.4 (PBS), containing 2 mM MgCI2, 0.5% BSA and 0.02 % NaN3 with or without nonradioactive peptides, monoclonal antibodies, or some soluble ligands for CD1 lb/CDI 8, and incubated for 1 h at 37°C. After washing the wells for three times with the binding buffer, the attached ~25I-labeled PI was solubilized with 1% SDS and counted. Saturation binding of peptide P1 to purified CD1 lb/CD18 was analyzed in dose response experiments in which increasing amounts of nonradioactive P1 were incubated in CDllb/CDl8-coated wells together with 12 pmol of 125I-labeled P1. Nonspecific binding was defined as the amount of 125I-P1 bound to CDI Ib/CD18 in the presence of a 100-fold excess of unlabeled PI, and was substracted from the total to calculate specific binding.

Binding of Endothelial Cells to Purified CDllc/CD18
Purified CD1 lc/CDI8 was diluted 1:10 and coated on flat-bottomed, 96-well microtiter plates, and the plates were saturated with 1% BSA as described above. The control plates were treated with BSA only. TNF-t~-stimulated Eahy926 cells were removed from the tissue culture flasks with 5 mM EDTA in PBS, washed, and resuspended in DMEM, 40 mM Hepes, pH 7.2, 2 mM MgCI2 and 5% FCS. Cells (5 × 104) in 50/~1 were added to each well in the absence or presence of 50/~g/ml of mAb 3.9 or control antibody MIgG, and incubated for 1 h at 37°C. Unbound cells were removed by gentle washing. The binding was quantitated by counting bound cells using 200× magnification of four randomly chosen fields from each well.

Aggregation Assays
Cells were washed with RPMI 1640 medium containing 40 mM Hepes, 2 mM MgCI2 and 2 mM CaCI2 and resuspended to a concentration of 106 cells/ml. Aliquots of 100 #l were added to each well of flat-bottomed, 96well microtiter plates in the absence or presence of peptides, and incubated at 37 °C for appropriate time periods. For inhibition of the ICAM-2 peptide Pl-induced cell aggregation, cells were preincubated with different monoclonal antibodies or inhibitors for 15 min at room temperature before being treated with the peptide. For quantitative measurement of cell aggregation, the free cells of four randomly chosen areas (2.5 mm 2) per well were counted. The amount of aggregated cells was expressed as: percent aggregation = 100 × [1-(number of free cells)/(total number of cells)].

Flow Cytometry Studies
THP-I and U937 cells were washed and resuspended in PBS, and treated with preimmune rabbit immunoglobulin to block the Fc receptors on the cells. Aliquots of 100 ttl of cell suspensions (106 cells) were incubated with 25/~g/ml of different mAbs for 30 min at 0°C. The cells were washed and incubated with FITC-conjugated rabbit-anti-mouse F(ab')2 (Dako, Copenhagen, Denmark) for 30 rain on ice. After washing, the cells were fixed with 1% paraformaldebyde in PBS, and 5 x 103 cells were analyzed immediately with a Becton-Dickinson (Immunocytometry System, San Jose, CA) FACScan flow cytometer.

Binding of THP-I Cells to Fibrinogen or iC3b Coated on Plastic
96-well microtiter plates were coated with purified fibrinogen (30 #g/ml in PBS), iC3b (2 #g/ml in PBS), or C3b (2 ~g/ml in PBS) for 16 h at 4°C. At the end of incubation, the wells were blocked with 1% BSA in PBS for 2 h at room temperature. 100-/zl aliquots of ADP or ICAM-2 peptidestimulated THP-1 cells at 2 x 106/ml in RPMI 1640 medium supplemented with 40 mM Hepes, 2 mM MgCI2, 2 mM CaCI2 were added to each well, and incubated for 30 min at room temperature. For blocking ex-periments, the peptide-stimulated cells were pretreated with different mAbs for 10 rain at 22°C before being added to fibrinogen, iC3b-, or C3b-coated wells. Nonadherent cells were removed by three washes with the binding medium. The binding was quantitated by scoring the number of attached cells with 200× magnification of four randomly chosen fields from each well.

Binding of lCAM-2 Peptide to CDllb/CD18
The purified CDllb/CD18 and CDllc/CD18 preparations were checked by polyacrylamide gel electrophoresis in the presence of SDS. The preparations contained the expected CD1 lb, CD1 lc, and CD18 polypeptides, and no major impurities were observed (Fig. 1 A, a and b). Most of the purified integrins was in functional intact heterodimeric forms as examined by running the preparations through the affinity columns once again, and checking the eluates by SDS-PAGE ( Fig. 1 A, c and d). The ~25I-labeled ICAM-2 peptide P1 bound to purified CD1 lb/CD18 coated on plastic, while little binding was observed to the purified CDllc/CD18 or BSA. The binding of J25I-labeled P1 to CDllb/CD18 was specifically and almost totally inhibited by unlabeled P1 (Fig. 2 A). The control peptide P8 had no effect. The purified CD1 lc/ CD18 was in active form as shown by the fact that Eahy926 cells bound to CDllc/CD18 coated on plastic, and the binding was blocked by the CDllc antibody 3.9 (Fig. 2 B). The binding of t25I-labeled P1 to CDllb/CD18 was saturable ( Fig. 3 A), and efficiently blocked by the anti-CD1 lb mAb   Figure 2. Binding of t2SI-labeled P1 peptide to purified CDllb/ CD18 and CDllc/CD18, and binding of Eahy926 cells to CDIlc/ CD18. 12 pmol of n25I-labeled P1 was incubated in CDIlb/CD18 or CD1 lc/CD18-coated wells in the absence or presence of 100/~M of nonradioactive P1 or P8 peptides (A). 50 #g/ml of mAb 3.9 and control antibody MIgG was used to block the adhesion of Eahy926 cells to CD1 lc/CDI 8 coated on plastic (B). The standard deviations are shown. The inhibitory effects of competing peptide P1 and mAb 3.9 were significant. *** p < 0.001.

ICAM-2 Peptide Pl-induced CDllb/CD18-ICAM-l-dependent Cell Aggregation
The kinetics of THP-1 and U937 cell aggregation induced by the peptide P1 was similar ( Fig. 4 A and Fig. 5 A). After 0.5-1 h incubation with P1, the cells clearly aggregated as compared to cells treated with the control peptide P8, or cells left without any treatment. Maximal aggregation was observed after 3-4 h incubation, and the cells remained in clusters during the experiments. Peptide P1 induced cell aggregation in a concentration-dependent manner, and a 50% aggregation was obtained with '~30 or 40/xM of P1 for THP-1 or U937 cells, respectively (Fig. 4 B and Fig. 5 B). The inhibitory effect of mAb OKMI0 was significant. ** p < 0.01. and U937 cells was studied by flow cytometry. CD18, CDllb, CDllc, ICAM-1, ICAM-2, and ICAM-3 were well expressed on both THP-1 and U937 cells, while little expression of CDlla was found (Fig. 6). Monoclonal antibodies against CD18 and ICAM-1 efficiently blocked the homotypic adhesion of both THP-1 and U937 cells, whereas the anti-CDllb antibodies OKM1, LM2/1, and 60.1 (for U937) blocked to a smaller, but significant extent (Fig. 4 C and Fig.  5 C). The CDllb antibody OKM10 and antibodies against CDlla, CDllc, ICAM-2, and ICAM-3 did not inhibit the aggregation.

Temperature, Energy, Microfilament, and Divalent Cation Requirements for ICAM-2 Peptide Pl-induced Cell Aggregation
No Pl-stimulated homotypic adhesion of THP-1 and U937 cells was detected at 4°C (Fig. 7, A and B). The aggregation was partially blocked by NAN3, but completely inhibited by using NaN3 in combination with 2-deoxy-o-glucose. Cytochalasin B, which prevents the formation of microfilaments, partially inhibited the aggregation, and EDTA blocked effi- ciently. Treatment of cells with dibutyryl cAMP, a cellpermeable cAMP analogue, slightly blocked the homotypic cell adhesion of THP-1 cells, but had no effect on U937 cells.

ICAM-2 Peptide Pl-induced Adhesion of THP-1 Cells to Fibrinogen
The purified fibrinogen was checked by polyacrylamide gel electrophoresis in the presence of SDS and found to be essentially pure (Fig. 1 B). THP-1 cells stimulated with the peptide P1 bound much more efficiently to fibrinogen coated on plastic (Fig. 8, A, B, and C a) than cells pretreated with the control peptide P8 (Fig. 8, A, B, and Cb) or cells left without any treatment (Fig. 8, A and B). ADP treatment also increased the binding of THP-1 cells to fibrinogen, but to a smaller extent (Fig. 8 A). 1 #M of P1 induced a substantial binding of THP-1 cells to fibrinogen, and the adhesion increased with increasing amounts of the P1 peptide (Fig. 8 B). The Pl-induced adhesion of THP-1 cells to fibrinogen was efficiently blocked by monoclonai antibodies to CD18 (7E4) and CD1 lb (OKM10), partially blocked by the CD1 lc antibodies 3.9 and 2El, and totally inhibited when antibodies OKM10 and 3.9 were used together (Fig. 9). The epitopes on CDllb/CD18 recognized by the monoclonal antibodies OKM1, OKM10, LM2/1, and 60.1 were expressed at similar levels on THP-1 cells (Fig. 6, data not shown). However, with the exception of OKM10, the other CD1 lb antibodies OKM1, LM2/1, and 60.1 did not block the binding. Another integrin receptor for fibrinogen, e¢4~3, is also expressed on THP-1 cells (data not shown), but the antibody LM609 reacting with this receptor was not inhibitory. The antibody to CD1 la (TS1/22), and the control antibody had no effect. EDTA abolished the adhesion completely.

ICAM-2 Peptide Pl-induced Adhesion of THP-1 Cells to Complement Fragment iC3b
The purified iC3b preparation was checked by polyacrylamide gel electrophoresis in the presence of SDS. It contained the ~ chain and the typical ot chain fragments, and no major impurities were observed (Fig. 1 B). Nonstimulated THP-1 cells bound to iC3b coated on plastic, but ADP and P1 peptide increased the binding to a small but significant extent, whereas the control peptide P8 had no effect (Fig. 10  A). The binding was efficiently inhibited by mAb 7E4, partially by OKM10 and 3.9, and an additive effect was observed when OKM10 and 3.9 were used together ( Fig. 10 B). Antibodies TS1/22, OKM1, LM2/1, and 60.1 were not inhibitory, whereas EDTA blocked the interaction. Little binding occurred to C3b.
Obviously, it is important to define the leukocyte integrin ligands in detail, determine the binding sites, and establish the mode of activation. ICAM-2 is a relatively simple integrin ligand, and therefore we thought that it should be an ex- We previously showed that the 22-amino acid residue peptide P1, derived from the first immunoglobulin domain of ICAM-2, specifically binds to CDlla/CD18 (Li et al., 1993b). However, we did not find any CDlla antibody that blocked the interaction. The P1 peptide inhibits the binding of endothelial cells to purified CD1 la/CD18, and the binding of lymphoblastoid cells to endothelial cells (Li et al., 1993b). In the present study, we show that the same peptide also specifically binds to purified CDllb/CD18, but not to THP-1 U937 . | O" "'"I"~I" ""+i'll2" "'ir~ . "''i'~N A CDlla CD11b i CD11c 3i i

ICAM-2 :
ICAM-3 ~)o" ""i~)U " "i~2" """ t"~ 3.''i~ ~ 16 °. "11i3 ~ . " "i+~" r 41i1+~ ' " " "il+~ . ~i++ '"i73c 'ion 'ii~+ " i~ ~ CDllc/CD18. The anti-CDllb mAb, OKM10, which recognizes a discontinuous epitope that requires the presence of both the NH2-terminal and divalent cation-binding regions , efficiently blocked this interaction. The results suggest that the epitope recognized by mAb OKM10 on the c~ chain is in the vicinity of the binding site for the P1 peptide. However, the soluble ligands for CD1 lb/ CD18 fibrinogen, iC3b, and factor X, were not able to interfere with this interaction, indicating that different regions on CD1 lb/CD18 are involved in binding of P1 and these ligands. The binding of the ICAM-2 derived peptide to CD1 lb/CD18 raises the interesting possibility that ICAM-2 could bind to this integrin. It has been shown that there exists a second CD1 lb/CD18 ligand on vascular endothelium, besides ICAM-1 (Carlos and Harlan, 1994). Our preliminary results indicate that ICAM-2 binds to CDllb/CD18 (Xie, J., R. Li, P. Kotou, C. Kantor, C. Vermot-Desroches, J. Wijdenes, M. A. Arnaout, P. Natamo, and C. G. Gahmberg, manuscript in preparation). The peptide P1 is relatively hydrophobic, and most part of it is probably buried in the first domain of ICAM-2, except that both of its NILE-and COOH-terminais seem to be exposed (Li et al., 1993b). It is essential that the peptide is in reduced form. We have found that when preserved without the presence of reducing agents, the peptide was easily oxi- dized and lost activity. On the other hand, alkylated peptide did not work either (data not shown). Therefore, the two cysteines in the peptide seem to be essential for its activity.
Recently, we found that the ICAM-2 peptide strongly induces blood T cell aggregation, which is mainly mediated by CDlla/CD18-ICAM-1 interaction, and an increase in the binding and cytotoxicity of natural killer cells (Li et al., 1993a). Here we show that the stimulatory effect is not restricted to T lymphocytes and natural killer cells. Myelomonocytic THP-1 and U937 cells became aggregated after treatment with the P1 peptide. Blood neutrophils were also strongly aggregated by P1 stimulation (data not shown). Unlike T lymphocytes, on which CDlla/CD18 is the main leukocyte integrin expressed, CDllb/CD18 and CDllc/CD18 are well expressed on THP-1 and U937 cells, while there is little expression of CDlla/CD18. The THP-1 and U937 cell aggregations were efficiently blocked by CD18 and ICAM-1 antibodies. The CDllb antibodies OKM1, LM2/1, and 60.1 (for U937) blocked to a smaller, but significant extent, while CD1 la and CD1 lc antibodies had no effect. These results in- Figure 8. Peptide Pl-induced adhesion of THP-I cells to fibrinogen coated on plastic. (,4) THP-1 cells were preincubated with 40 #M of peptides P1 or P8 for 2 h at 37°C, washed, and resuspended by pipetting, before being added to fibrinogen or BSA-coated wells. 10 #M of ADP was added at the same time when the cell suspension was added to the wells. (B) THP-1 cells were stimulated with different concentrations of PI (u) or P8 (o), or left without any treatment (A), before being added to fibrinogen-coated wells. (C) Binding of peptide P1 (C a) or P8 (C b)-stimulated THP-I cells to immobilized fibrinogen. The photomicrographs were taken after the nonadherent cells had been removed by washing three times. A 200x magnifcation was used. The bar indicates 50/~m. dicate that CDllb/CD18 becomes activated, and mediates the cell aggregation by interacting with ICAM-1. Since the CDllb antibody OKM10, which blocked the binding of P1 peptide to purified CDllb/CD18, did not inhibit the THP-1 and U937 cell aggregation, it is probable that CDIlb/CD18 is activated by signals resulting from the interaction of P1 with the small amount of CDlla/CD18 expressed on the Aliquots of Pl-stimulated THP-1 cells were separately pretreated with 50 /.tg/ml of mAbs against CD18 (7E4), CDlla (TS1/22), CDIlb (OKM1, OKM10, LM2/1, and 60.1), CDllc (3.9, 2El), avfl3 (LM609) and control MIgG1, before being added to fibrinogen-coated wells. 5 mM EDTA was used. The effect of P1 is significant as indicated.
cells. Alternatively, during the incubation of the cells at 37°C, the OKM10-engaged CDllb/CD18 molecules are no longer available on the cell surface, because of the rapid internalization of occupied CD1 lb/CD18 (Rab et al., 1993). It has been shown that translocation of neutrophil CD1 lb/CD18 complex to the cell surface can be induced at 37°C (Todd et al., 1984). We think that a minor possibility could be that these OKM10-free CD1 lb/CD18 derived from the intracellular pool bound P1 and became activated. Peptide Pl-induced homotypic adhesion of THP-1 and U937 cells does not happen at 4°C, and requires energy, intact microfilaments, and divalent cations. However, cytochalasin B, which prevents the formation of microfilaments, could not totally block the aggregation; and CD18 antibody 7E4 was not able to completely inhibit the aggregation either. These findings suggested that besides f12 integrins, some other integrins or adhesion systems were also activated.
We previously reported that cross-linking of CD1 lb/CDI 8 on neutrophils with monoclonal antibodies causes a rise in cytosolic free Ca 2+, which is functionally coupled to a transient activation state of CDllb/CD18 (Altieri et al., 1992). In addition, engagement of the divalent ion binding site(s) on CD1 lb/CD18 induces the expression of activation-dependent neoantigenic epitopes on CDllb/CD18 and leukocyte adhesion (Altieri, 1991). Furthermore, binding of eosinophils to endothelial cells causes a considerable upregulation of CDllb and an increased capacity to generate an oxidative burst (Walker et al., 1993). It is possible that binding of P1 to CDllb/CD18 has a similar effect as CDllb-specific activating antibodies. The complexity of integrin-activating antibodies is illustrated by the opposite effects of CD1 la and CD18 antibodies on T cell activation (van Noesel et al., 1988). In the presence of CD18 antibodies, CD1 la antibodies increased T cell proliferation, whereas CD18 antibodies were inhibitory. These findings indicate that integrin-ligand Figure 10. Peptide Pl-induced adhesion of THP-1 cells to iC3b or C3b coated on plastic. (A) THP-1 cells were preincubated with 40 /xM of peptides P1 or P8 for 2 h at 37°C, washed, resuspended by pipetting, and added to iC3b-or C3b-coated wells. 10/xM of ADP was added at the same time when the cell suspension was added to the wells. (B) Aliquots of Pl-stimulated THP-1 cells were separately pretreated with 50 /~g/ml of mAbs against CDI8 (7E4), CDI la (TS1/22), CD1 lb (OKM1, OKM10, LM2/I, and 60.1), CD1 lc (3.9), and control MIgGI, before being added to iC3b-coated wells. 5 mM EDTA was used.
binding may have different effects on integrin functions depending on the site of interaction.
Although several stimuli such as phorbol esters, the peptide formyl-methionyl-leucyl-phenylalanine and complement fragment C5a, can increase the cell surface expression of CDllb/CD18 (Lo et al., 1989;Detmers et al., 1990;, no detectable increase in CD1 lb/ CD18 expression on granulocytes and THP-I cells was observed after treatment with P1 (data not shown). Phorbol ester or ADP-induced high avidity state of CDllb/CD18 is characterized by the formation of activation-dependent neoantigenic epitopes recognized by the ct chain antibodies CBRM1/5  or 7E3 (Altieri and Edgington, 1988b). However, the P1 peptide-stimulated functional modulation of CD1 lb/CD18 was not accompanied by an increased expression of these epitopes (data not shown). Recent work has shown by using CD18 antibodies, that the peptide P1 was able to induce the aggregation of the /32 integrins in the plane of cell membrane on natural killer cells (Somersalo et al., 1995), which is supposed to be tightly correlated with high ligand-binding capacity of CDllb/CD18 (Detmers et al., 1987).
The adhesion of THP-1 cells to fibrinogen and iC3b, was increased by stimulating the cells with peptide P1. The interactions were efficiently blocked by CD18, CD1 lb, and CD1 lc antibodies, and an additive effect was observed when CD1 lb and CD1 lc antibodies were used together. The Pl-stimulated adhesion of THP-1 cells to immobilized fibrinogen and iC3b was blocked by the antibody OKM10, which was found previously to be able to block the interactions between leukocytes and immobilized fibrinogen and iC3b (Wright et al., 1983(Wright et al., , 1988Anderson et al., 1986). On the other hand, some reports have shown that OKM1, rather than OKM10, inhibits the interaction between the leukocytes and soluble fibrinogen (Altieri et al., 1988(Altieri et al., , 1990. It has been found that conformational changes occur in fibrinogen when the protein is adsorbed onto a plastic surface (Ugarova et al., 1993). This could be the reason for the fact that soluble fibrinogen did not inhibit the interaction between P1 and CDllb/CD18, in which the OKM10 epitope was involved. The background binding of resting THP-1 cells to iC3b coated on plastic was quite high, because iC3b constitutes a ligand for nonactivated CDllb/CD18 (Ross and Vetvicka, 1993).
Taken together, our results are compatible with a model in which the ICAM-2-derived peptide PI binds to the leukocyte integrins CDlla/CD18 (Li et al., 1993b) and CDllb/ CD18, but not to CDllc/CD18. These interactions induce high avidity states of CDlla/CD18 on T lymphocytes (Li et al., 1993a), and CDllb/CD18 and CDllc/CD18 on monocytic cells for their ligands (Fig. 11). Evidently, the activation of CDllb/CD18 and CDllc/CD18 involves both outside-in and inside-out signals, but it is not clear yet whether the signals come from the interaction of P1 peptide with CDlla/ CD18, or with CDllb/CD18, or with both. The detailed mechanisms involved in the signaling remain poorly understood.
We tested the ability of peptide P1 to induce the binding of CDllb/CD18 transfected CHO cells to fibrinogen. The CDllb and CD18 are in the expression vector rH3M (Dana et al., 1991), and was obtained from Dr. M. A. Arnaout. Approximately 20-30% of the transfected cells expressed CDllb/CD18 as determined by FACScan analysis. The CDllb/CD18 transfected cells bound to fibrinogen, but no additional activation by P1 was observed (data not shown). Phorbol esters were not either capable of activating the transfected cells. These findings show that the CD1 lb/CD18 is active in the transfected cells, but further activation is difficult to achieve. Evidently, CHO cells lack the activation system found in leukocytes.
Recent studies have provided evidence of how fibrinogen participates in inflammatory responses and host defense. Fibrinogen is involved in the regulation of leukocyte adhesion to vascular endothelium by interacting with both CDllb/CD18 on leukocytes and ICAM-1 on endothelium (Altieri et al., 1993;Languino et al., 1993). The finding that P1 stimulates the binding of leukocytes to fibrinogen raises the intriguing possibility that ICAM-2, which is constitutively expressed on most endothelia (Nortamo et al., 1991a;de Fougerolles et al., 1991), could have an important regulatory role in leukocyte binding during physiological conditions.
Besides the proposed triggering role for ICAM-2 peptide Figure 11. Hypothetical model to explain the ability of the ICAM-2-derived peptide P1 to regulate the avidity of leukocyte integrins. Peptide P1 binds to CDlla/CD18 and CDllb/CD18, but not to CDllc/CD18. The binding induces outside-in and inside-out signals, by which CDlla/CD18 (Li et al., 1993a), CDllb/CD18, and CDllc/CDI8 are activated to bind to their ligands.
P1 in ligand binding to integrin, it is obvious that this peptide also contributes to the adhesive interaction itself. This is based on the capacity of P1 to bind to purified CD1 la/CD18 and CDllb/CD18, as well as the capacity of this peptide to inhibit the adhesion of endothelial cells to purified CD1 la/ CD18. Furthermore, it partially blocks the binding of B lymphoblastoid cells to endothelial cells (Li et al., 1993b). The ability of P1, on one hand, to inhibit the binding of ICAM-2 containing nonleukocytic cells to CD1 la/CD18, but on the other hand, to activate leukocyte binding, is incompletely understood. The main reason may be that as shown here, the three CDll/CD18 cellular integrins become activated by P1, and they bind to several ligands at different binding sites. Therefore, blocking of activated cell-bound integrins is more difficult than interference with the binding of single purified integrin to its ligands. An explanation for the high avidity ligand binding triggered by peptide P1 could be that additional ligand-binding sites are generated in the integrin, as proposed for the activation of the platelet integrin gplIb/IIIa by arginine-glycineaspartic acid (RGD)-containing peptides Pierschbacher, 1986, 1987;Du et al., 1991). However, unlike the RGD-containing peptide-induced high affinity state of gplIb/IIIa, which appears to be due to a direct conformational change in the receptor itself (Duet al., 1991), the P1induced activation of B2 integrins seems to involve signal transduction events (Li et al., 1993a), although a direct effect on CDll/CD18 integrins cannot be excluded.
Our results show that the high avidity state of leukocyte integrins for different ligands can be induced by interaction between the integrins and the ICAM-2-derived P1 peptide. This indicates the possibility of induction of active forms of leukocyte integrins through interaction with agonistic ligands. Such regulatory reagents could be useful in therapeutical maneuvers in the future.
We thank Veronica Ehrnrooth and Yvonne Heinil~i for expert secretarial assistance, Leena Kuoppasalmi for technical assistance, Dr. T. A. Springer for the CDlla antibody TS1/22, Dr. N. Hogg for the CDllc antibody 3.9, Dr. C. Vermot-Desroches for the ICAM-2 antibody B-T1, Dr. D. L. Simmons for the ICAM-3 antibody CG106,, Dr. Jari Yl~inne for the avfl3 antibody LM609 and purified fibrinogen, and Dr. M. A. Arnaout for the CD1 lb and CD18 containing expression vectors.