©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Natural Killer Cell Migration by Leukocyte Integrin-binding Peptide from Intracellular Adhesion Molecule-2 (ICAM-2) (*)

Kristina Somersalo (1)(§), Olli Carpén (1), Eero Saksela (1), Carl G. Gahmberg (2), Pekka Nortamo (2), Tuomo Timonen (1)

From the (1) Department of Pathology and the (2) Department of Biochemistry, University of Helsinki, FIN-00014 Helsinki, Finland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Intracellular adhesion molecule-2 (ICAM-2), one of the ligands of CD11a/CD18 (LFA-1), is mainly expressed on endothelial and hematopoietic cells. The biological significance of ICAM-2 has remained unclear. Previous findings have shown that a peptide from ICAM-2, spanning residues 21-42 from the first immunoglobulin domain, enhances natural killer (NK) cell cytotoxicity and induces T cell aggregation. We have now studied the effect of the same ICAM-2 peptide on NK cell migration in the Boyden chamber assay. The peptide significantly increased NK cell migration up to 215 ± 21%, as compared to migration of control cells (100%), and the induction was inhibited by anti-CD11a monoclonal antibodies. The ICAM-2 peptide also induced polymerization of F-actin at the leading edge of migratory NK cells. Cross-linking of CD11a/CD18 receptors with anti-CD11a or anti-CD18 monoclonal antibodies and secondary antibodies resulted in receptor recycling, increased migration, and actin polymerization, but led to slight inhibition of cytotoxicity. The ICAM-2 peptide did not induce such a receptor recycling. Phosphotyrosine immunoblotting experiments showed that the ICAM-2 peptide increased the phosphorylation of 150- and 35-kDa proteins. During cross-linking with antibodies, only the 150-kDa protein showed increased phosphorylation. The results show that depending on the type of CD11a/CD18 receptor ligation different kinds of signals are transduced in NK cells. These signals may either trigger only locomotion, or both locomotion and cytotoxicity. Based on these findings, a major function for ICAM-2 on endothelium may be triggering of migration of adhering leukocytes.


INTRODUCTION

NK() activity represents a form of early defense mechanisms (1, 2) . While normally found mostly in blood and spleen, NK cells accumulate to sites of microbial infections, tumors (1, 2) , and rejecting allografts (3, 4) . The trafficking of NK cells to strategic sites involves the ability to respond either to soluble or surface-bound signals that stimulate cell migration. When a lymphocyte is triggered to locomote, its initial carbohydrate-mediated loose binding on endothelium is followed by firm integrin-mediated adhesion (5) . Thereafter, the lymphocyte spreads on endothelium, and subsequent transmigration is associated with the polymerization of actin to the leading edge of a cell, as a result of receptor-mediated signal transduction (6) .

Many factors that attract leukocytes at low concentrations also increase cytotoxicity at higher concentrations (6) . This is biologically meaningful because the highest concentration of a factor exists in the actual lesion. When confronting the target cell, the NK cell stops its migration, adheres to the target mostly via -integrins, and the cytolytic mechanism is triggered: the Golgi apparatus, the centrioles, granules, and polymerized actin reorient toward the contact area (2, 7, 8) . In addition, cytoplasmic calcium increase, inositol turnover, and protein kinase activation are associated with the function of the cytotoxic machinery (2) .

Adhesion receptors play a pivotal role in inflammatory responses. To be able to locomote, a cell must attach to other cells or to the extracellular matrix, and cytotoxicity requires a tight and transient adhesion of the effector cell to the target. CD11a/CD18 (LFA-1) is the major integrin of lymphocytes. It is a heterodimer and composed of noncovalently bound - (CD18) and -subunits (CD11a). In resting circulating lymphocytes, CD11a/CD18 is in a nonadhesive conformation, and in order to become adhesive it requires triggering by an external signal. Certain anti-CD11a/CD18 mAbs (9, 10) , divalent cations (11, 12) , or a newly found lipid (IMF-1) (13) , can directly induce an adhesive conformation to this receptor. The avidity increase of CD11a/CD18 is also induced by signaling through certain chemotactic receptors (14, 15, 16) through CD3 cross-linking (17) and through the E-selectin ligand (18) . In stimulated NK cells, the high avidity state CD11a/CD18 receptors participate in many adhesion-dependent functions: binding to endothelial cells (19, 20) , migration (21, 22, 23) , and cytotoxicity (24) . In addition to binding, CD11a/CD18 may also function as a triggering receptor in the activation of migration and cytotoxicity. Cross-linking of TCR (25, 26, 27) on resting T cells results in both a high avidity state of CD11a/CD18 and rapid cell proliferation, when CD11a/CD18 is coligated. In addition, cross-linking of CD11a itself has been shown to be capable of signal transduction through calcium increase and inositol hydrolysis (28) .

Ligands for the CD11a/CD18 integrin are ICAM-1, ICAM-2, and ICAM-3. ICAM-1 differs from ICAM-2 in distribution and inducibility. Apart from mere adhesion very little is known about the biological roles of ICAMs. ICAM-1 is widely distributed, and its expression can be up-regulated by various cytokines. ICAM-2 is mainly present on vascular endothelium and lymphohematopoietic cells (29, 30, 31) , and it is generally constitutively expressed (29) although its expression may be increased in lymphoid malignancies (32) . A costimulatory cross-talk between TCR and ICAM-1 (33) or ICAM-2 (34) has been demonstrated. The ICAM-1 and ICAM-2 expression on target cells contributes to LAK cell killing sensitivity (35) but not to endogenous NK cell killing (36) .

We have previously shown that a soluble 22-amino-acid long peptide from the first immunoglobulin domain of ICAM-2, but not from the homologous region of ICAM-1 activates T cell aggregation and NK cytotoxicity (37) . In the present work we show that the same peptide induces actin polymerization, redistribution of actin, and increased migration of endogenous NK cells. In addition, we show that although receptor cross-linking with CD11a- or CD18-specific mAbs resulted in actin polymerization and increased migration, it did not affect cytotoxicity. The results indicate that CD11a/CD18 alone is capable of signal transduction and that the response to outside signals depends on ligand quality. Furthermore, our results suggest a biological role for ICAM-2 in activation of cell migration.


MATERIALS AND METHODS

Cell Purification

Buffy coats from healthy blood donors were obtained from the Finnish Red Cross Blood Transfusion Service. NK and T cells were isolated by Ficoll-Isopaque (Pharmacia Fine Chemicals AB, Uppsala, Sweden) gradient centrifugation, filtration through nylon wool columns, and by four-step Percoll (Pharmacia) gradient centrifugation in RPMI 1640 (Life Technologies, Inc.) supplemented with 0.29 mg/ml glutamine (Life Technologies, Inc.), 100 IU/ml penicillin, 10 µg/ml streptomycin, and 5% heat-inactivated fetal calf serum (Life Technologies, Inc.), as described previously (38) , with minor modifications. Purified lymphocytes were phenotyped by flow cytometry. Lymphocytes in the NK cell fraction consisted of 54.1 ± 9.6% of CD56+CD3-, 44.2 ± 11.7% of CD3+, and 1.3 ± 1.1% of CD14+ cells ( n = 39) (mean ± S.D.). Highly purified NK cells were used in immunofluorescence microscopy, immunoblotting, and phosphatidylinositol turnover experiments. They were obtained as described by Timonen et al. (39) : NK fraction cells from the Percoll gradients were incubated at 29 °C for 45 min with sheep red blood cells at a ratio of 1:150. The sheep red blood cells with the attached CD3+ cells were removed by a subsequent Ficoll-Isopaque gradient centrifugation. The resulting cell populations contained 90.1 ± 4.8% CD56+CD3-, 4.2 ± 1.8% CD3+, and 0.3 ± 0.4% CD14+ lymphocytes.

Antibodies and Phenotypic Analysis

The mAbs used in migration, cytotoxicity, and phosphatidylinositol (IP) assays were: anti-CD11a (IOT16, IgG(40) , Serotec, Bicester, Oxon, UK), anti-CD18 (7E4 (41) purified IgG, or IB4, IgG2a, courtesy of Dr. Samuel Wright, Rockefeller University, New York), anti-CD16 (3G8 (42) purified IgGfrom supernatant of hybridoma cell line), and F(ab`)fragments of goat-anti-mouse-IgG (Coulter Immunology, Hialeah, FL) as secondary cross-linking antibody. The mAbs used in phenotyping were: anti-CD16, anti-CD56, anti-CD3, and anti-CD14 (anti-Leu11b, anti-Leu19, anti-Leu4, and LeuM3, respectively, all from Becton-Dickinson, Mountain View, CA). Normal mouse IgG, including all subclasses (Coulter Immunology), was used as a control. Phenotypic analysis was carried out using standard procedures (23) , and the cells were analyzed using a FACScan (Becton-Dickinson) flow cytometer.

Peptides

A 22 amino peptide (residues 21-42) from the first immunoglobulin domain of ICAM-2 (GSLEVNCSTTCNQPEVGGLETSL), and a control peptide containing the same amino acids as the ICAM-2 peptide, but in random order, except for the two conserved cysteines (EVGTGSCNLECVSTNPLSTGTEQ) were used (43) . Additionally, ICAM-1 (GSVLVTCSTSCDQPKLLGIETPL) and ICAM-3 (GSLFVNCSTDCPSSEKIALETSL) peptides made from the homologous regions were used. The peptides were synthesized by t-Boc chemistry on an Applied Biosystems 430A peptide synthesizer and purified to >98% purity by high performance liquid chromatography. The quality of the purified peptides was verified by amino acid sequence analysis and mass spectrometry (43) .

Cell Activation

The soluble ICAM-2 peptide or the soluble control peptide (0-100 µg/10 10cells/ml) was added to lymphocyte suspensions 30 min before experiments and left in the supernatants during the assay. Activating mAbs (anti-CD18 (7E4 or IB4), anti-CD11a (IOT16), or anti-CD16 (3G8)) were added (10 µg/1 10cells/100 µl) at 4 °C 30 min before experiments and left in the supernatants during the assay. In cross-linking experiments, the cells were first incubated with a primary mouse mAb for 30 min at 4 °C, washed, and F(ab`)fragments of goat-anti-mouse-IgG were added, incubated for 30 min at 4 °C, and left in the supernatants during the assay.

Migration Assay

The Boyden chamber migration assay described by Axelsson et al. (44) was used. Polycarbonate filters with pores of 3-µm diameter (Nuclepore Corporation, Pleansanton, NY) were used between the lower and upper compartments of the Boyden chambers. The lower compartments were filled with 400 µl of RPMI 1640 supplemented with 0.5% human AB serum and 0.5% human serum albumin (both from Finnish Red Cross Blood Transfusion Service). The Percoll fractionated lymphocytes were suspended in the same medium at 10 10/ml and activated as described above. 200 µl of lymphocyte suspensions were added to the upper compartments. As a background control to monitor passive relocation of cells to the lower compartment of the chamber, the divalent cation chelator EDTA (10 m M), which inhibits migration was used (23) . After incubation for indicated hours at 37 °C, the amount of migratory cells in the lower chambers was counted. NK or T cell frequencies were determined by phenotyping, the amounts of migrated NK cells were calculated, and the mean values in triplicate chambers were calculated. The cell number of the EDTA-treated cells (<5% of input) in the lower chamber was subtracted from the experimental cell numbers. In some experiments the cells were treated with various concentrations of genistein (Life Technologies, Inc.), staurosporin (Sigma), or okadaic acid (Sigma) for 30 min at 37 °C before the assays and left in the supernatants during the assay. Genistein and staurosporin were dissolved in MeSO and okadaic acid in ethanol. MeSO and ethanol at their final concentrations used had no effect on the parameters determined. The results are expressed as the mean ( ± S.E.) values of repeated experiments.

Cytotoxicity Assays

1 10K562 target cells (45) (American Type Culture Collection, Rockville, MD) were labeled with 20 µCi of sodium [Cr] (Radiochemical Centre, Amersham, United Kingdom). Various concentrations of effector cells were added in 100 µl to 100 µl of target cells 1 10/ml, to produce effector/target ratios 20:1, 10:1, 5:1, and 2.5:1. After a 4-h incubation at 37 °C, 100 µl of the supernatant from each well was counted in a gamma counter (Wallac, Turku, Finland). The percentage of radioactivity released was calculated as follows:

On-line formulae not verified for accuracy

 

On-line formulae not verified for accuracy

Fluorescence Microscopy

Highly purified NK cells were used. For visualization of filamentous F-actin, activated NK cells were fixed in 3.5% paraformaldehyde for 10 min, washed with phosphate-buffered saline, and permeabilized with 0.1% Triton X-100 for 5 min. The cells were then incubated with tetrametylrhodamine isothiocyanate (TRITC)-conjugated phallacidin (Molecular Probes Inc. Pitchford, TX) which binds to F-actin. In cross-linking experiments, unspecific Fc binding was first blocked with 20% human AB serum. The cells were incubated with the primary mAb (anti-CD18 (7E4) or anti-CD11a (IOT16)) for 30 min at 4 °C, washed, and further incubated with F(ab`)fragments of goat-anti-mouse-IgG for 30 min at 4 °C, then further incubated at 37 °C for various times (0-3 h), washed, and stained with the FITC-labeled rabbit-anti-goat-IgG (Cappel, Organon Teknika Corp., Durham, NC) for 30 min at 4 °C. When indicated, the cells were permeabilized with 0.1% Triton X-100 for 5 min and then stained with anti-CD11a (N225, N226, and N217 obtained through the Fourth International Leukocyte Workshop), anti-CD18 (7E4), and the secondary FITC-labeled goat-anti-mouse-IgG for the detection of cytoplasmic CD11a/CD18. After washing, the cells were and fixed in 3.5% paraformaldehyde.

SDS-polyacrylmide Gel Electrophoresis and Immunoblotting

Highly purified NK cells were used. 10 10NK cells were lysed in 50 m M HEPES, pH 7.2, 1% Triton X-100, 0.1% SDS, 10 m M EDTA, 100 m M NaHPO, 10 m M NaF, 1 m M NaVO, and 10 µg/ml aprotinin and leupeptin. The soluble proteins were mixed in Laemmli buffer, separated in 8% polyacrylamide gels under reducing conditions (46) , and subsequently transferred to nitrocellulose filters. The filters were blocked with 4% bovine serum albumin overnight, incubated with mouse-anti-phosphotyrosine (ascites fluid 1:1000) (Sigma) for 1 h, and exposed to peroxidase-conjugated rabbit-anti-mouse IgG (Dako A/S, Glostrup, Denmark) for 30 min. Bound antibody was visualized by ECL (Amersham) according to the manufacturer's instructions.

Phosphatidylinositol Experiments

The procedure described by Stand Carpén (47) was used. Activated highly purified NK cells were incubated (2.5 10cells/ml) in inositol-free culture medium (RPMI 1640 sine inositol, Pharmacy of Helsinki University, Finland), containing 0.5% of human serum albumin, for 24 h together with [H]inositol 35 µCi/ml (NET 906, DuPont NEN). After washing, the cells were suspended (20 10cells/ml) in inositol-free culture medium containing 0.5% human serum albumin and 10 m M LiCl in order to inhibit the activity of inositol phosphate phosphatase. The cells were then activated with indicated agents for 3 min at 37 °C. The inositol phosphate metabolism was terminated by the addition of methanol/chloroform (2:1) and thereafter by chloroform/water (1:1) to the cell suspensions. The water-soluble inositol phosphates were separated and fractionated on MonoQ anion-exchange (fast protein liquid chromatography) system (Pharmacia, Uppsala, Sweden) with a continuous gradient of 0-0.1 M ammonium formate. One-ml fractions were collected, and the radioactivity was counted with a -counter (LKB-Wallac, Bromma, Sweden).

Statistical Methods

The statistical significance of the results in cell migration assays was evaluated by the paired t test (* = p < 0.001, = 0.001 < p < 0.01, * = 0.01 < p < 0.05).


RESULTS

Effect of the ICAM-2 Peptide on NK Cell Migration

To extend the formerly described experiment, where the ICAM-2 peptide was shown to stimulate NK cell binding and cytotoxicity (37) , the effect of increasing concentrations of the soluble ICAM-2 peptide or control peptide on the migratory capacity of NK cells was examined. Freshly isolated and ICAM-2 peptide-treated peripheral blood lymphocytes were seeded on the top of filters dividing Boyden chambers into two compartments. The ability of NK cells to migrate through the filters into the lower compartments of the chamber during 4 h was determined. As shown in Fig. 1, the NK cell migration was significantly increased by the ICAM-2 peptide. The migration was dependent on the concentration of the peptide, and the plateau response was reached at 75 µg/ml. The increase of migration was inhibited by anti-CD11a (IOT16 10 µg/1 10cell/100 µl) mAb (not shown) although the random migration was not dependent on CD11a. ICAM-1, ICAM-3, and control peptides had no effect on the migration (not shown). In kinetic experiments, significantly increased NK cell migration was first detectable after a 4-h treatment with the ICAM-2 peptide (Fig. 2).

The Effect of the ICAM-2 Peptide on F-actin Distribution and the Spreading of NK Cells

Cell movement involves polymerization of globular actin into F-actin and its distribution toward the leading edge. We therefore wanted to examine the effect of the ICAM-2 peptide on actin polymerization in highly purified NK cells. Prior to stimulation the cells were round (Fig. 3 A), and F-actin visualized by TRITC-phallacidin was homogeneously distributed in the cytoplasm. After stimulation, all NK cells spread on the substratum and obtained the morphology of migratory cells with a leading edge and a trailing edge. F-actin staining in these cells became more abundant than in control cells, and often F-actin was localized in the leading edges of the cells (Fig. 3 B).


Figure 3: Analysis of the distribution of F-actin in NK cells. Highly purified CD56+/CD3- NK cells were activated by a control peptide ( A), ICAM-2 peptide (7.5 µg/1 10cell/100 µl) ( B), or by cross-linking of the CD11a (IOT16 mAbs followed by F(ab)fragments of goat-anti-mouse IgG Ab) ( C) or CD18 (7E4 mAbs 10 µg/1 10cell/100 µl and the secondary Ab) ( D). After fixation F-actin was visualized by rhodamine-phallacidin.



Antibody-induced cross-linking of CD11a and CD18 caused similar cell spreading and F-actin polarization as the ICAM-2 peptide did (Fig. 3, C and D). If CD11a/CD18 was not cross-linked with secondary antibody, no effect was seen (not shown).

Effect of Cross-linked CD11a/CD18 on Migration and Cytotoxicity

It is apparent that the induction of NK cell migration by the ICAM-2 peptide involves ligation of the CD11a/CD18 integrin. We wanted to find out whether cross-linking of CD11a or CD18 by specific mAbs would have a similar effect. As seen in Fig. 4, cross-linking of CD11a with IOT16 mAb and secondary Ab increased the migration as strongly as the soluble ICAM-2 peptide. The cross-linking of CD18 with 7E4 or IB4 mAb and a secondary Ab also increased NK cell migration, but less than the cross-linking of the -chains.

Because the ICAM-2 peptide is known to increase the cytotoxicity of NK cells (37) (Fig. 4), we also studied the effect of CD11a/CD18 cross-linking on cytotoxicity. Cross-linking (IOT16, 7E4, or IB4) slightly inhibited cytotoxicity during the 4-h assay, as seen in Fig. 4. The cytotoxicity did not increase even if the cells were preincubated for 4 h with primary and secondary mAbs before the experiments. CD18 has a role as an adhesion receptor in the binding and migration of NK cells (23, 48) . Therefore, when the IB4 epitope on CD18 antigens was covered with IB4 mAbs without cross-linking, both migration (23) and cytotoxicity (48) of endogenous NK cells were inhibited (Fig. 4). Control peptide or control mAb had no effect. Surprisingly, when the 7E4-binding site on CD18 was covered, without cross-linking, migration was inhibited but not cytotoxicity (not shown).


Figure 4: Effects of ICAM-2 peptide and antibody cross-linking on NK cell migration and cytotoxicity. Enriched NK cells were activated by ICAM-2 peptide (7.5 µg/1 10cell/100 µl) or by cross-linking of CD11a or CD18 receptors (IOT16 or IB4 mAbs 10 µg/1 10cell/100 µl and a secondary Ab). After a 4-h incubation at 37 °C, the migration of CD56+/CD3- NK cells was calculated or the cytotoxicity of NK cells against K562 cells was measured. The results are expressed as percent increase compared to untreated control cells. The average control migration was 120-200 10migrated cells/4 h incubation and control cytotoxicity 500-2000 cpm/4 h incubation. Mean values from four to eight experiments ± S.E. *** = p < 0.001, ** = 0.001 < p < 0.01, * = 0.01< p < 0.05.



Because cross-linking of CD18 with the mAbs (IB4 and 7E4), which cover the epitopes responsible for migration, increased migration we examined the effect of cross-linking on possible recycling of the CD11a/CD18 integrin and the re-expression of free integrin during the 3-h incubation. When the cells were treated with anti-CD11a (IOT16) or anti-CD18 (7E4) mAbs, the fluorescence intensity of the surface CD11a or CD18 molecules did not change during the 3-h incubation at 37 °C. If molecules were cross-linked with the secondary Ab, the intensity of the surface CD11a or CD18 decreased significantly (Fig. 5 shows CD18 expression). Immunofluorescence microscopy revealed that CD11a or CD18 started to patch immediately after the cross-linking, and after 3 h of incubation the receptors were completely capped in 98% of the cells. If the cells were permeabilized at this time point, restaining visualized the internalized CD11a and CD18 molecules, and surface restaining of CD11a or CD18 revealed the re-expression of free adhesion molecules, as seen in Fig. 5 F. The ICAM-2 peptide did not induce such a receptor recycling (not shown).

Role of Protein Phosphatases and Kinases on Stimulated Migration and Cytotoxicity

Experiments were performed to analyze possible differences between the two stimulation mechanisms, which in one case leads to increased migration and cytotoxicity (ICAM-2 peptide), and in the other to an increased migration but not to increased cytotoxicity (CD11a/CD18 cross-linking). We first studied whether different protein kinases and phosphatases participate in random uninduced migration and endogenous cytotoxicity of NK cells. Both the tyrosine protein kinase inhibitor genistein and the serine/threonine protein kinase inhibitor staurosporin decreased the random migration and cytotoxicity in a dose-dependent fashion (Fig. 6). Staurosporin abrogated the activities completely, whereas genistein inhibited migration by 49% and cytotoxicity by 59%. Okadaic acid is a specific protein phosphatase 2A inhibitor when used at lower concentrations than 10 n M, and a protein phosphatase 1 inhibitor when used at 10 n M-1 µ M (49) . It had no effect on migration at concentrations lower than 400 n M, but at a concentration of 1 µ M it decreased migration by 52%. In contrast, all concentrations of okadaic acid tested (from 150 n M to 1 µ M) increased endogenous cytotoxicity.

The effect of the ICAM-2 peptide and CD11a/CD18 cross-linking on NK cells in the presence of inhibitors was then studied. The inhibitors had similar effect on activated cells and on uninduced cells; moreover, migration induced by the ICAM-2 peptide or by CD11a/CD18 cross-linking were similarly dependent on the inhibitors (Table I).

Effect of the ICAM-2 Peptide and CD11a/CD18 Cross-linking on Tyrosine Phosphorylation

The results indicated that protein phosphorylation, among other effects, is required for migration and cytotoxicity. The possibility that activation of NK cells by the ICAM-2 peptide or CD11a/CD18 cross-linking by mAbs could lead to different patterns of protein phosphorylation was studied next. The detergent-soluble proteins of the ICAM-2 peptide-activated or CD11a/CD18 cross-linking-activated highly purified NK cells were probed with anti-phosphotyrosine antibodies. As seen in Fig. 7 A, the ICAM-2 peptide, which activated both migration and cytotoxicity, increased phosphorylation of two cellular proteins, of 150 and 35 kDa. The phosphorylation was detected most clearly after 8 and 15 min of activation. The control peptide did not cause any phosphorylation. Cross-linking with mAbs resulted in phosphorylation of the 150 kDa, but not the 35-kDa protein (Fig. 7 B).

Effect of the ICAM-2 Peptide and CD11a/CD18 Cross-linking on Phosphatidylinositol Turnover

Caincrease, a consequence of the hydrolysis of plasma membrane phosphoinositides, is involved in both cytotoxicity and migration. The effect of the ICAM-2 peptide and CD11a/CD18 cross-linking on the NK cell inositol turnover was therefore investigated. Fig. 8 shows that the ICAM-2 peptide increased only marginally the phosphatidylinositol monophosphate (IP1) accumulation. Cross-linking of CD11a/CD18 increased the IP1 accumulation significantly, although not as much as the positive control, a mAb to CD16. No increase was observed when NK cells were incubated with anti-CD11a or anti-CD18 mAbs without secondary antibodies.


DISCUSSION

Earlier studies have indicated that the CD11a/CD18 integrin binds to the first immunoglobulin domain of ICAM-1 (50) . The 6D5 mAb to ICAM-2 reacted with its first immunoglobulin domain and inhibited leukocyte adhesion to ICAM-2-transfected COS-1 cells indicating that this domain is most important in CD11a/CD18 binding (51) . Subsequent studies showed that a synthetic 22-amino-acid long peptide, covering residues 21-42 of the first immunoglobulin domain, inhibited binding of endothelial cells to purified CD11a/CD18 and to B lymphoblastoid cells (43) . The same soluble ICAM-2 peptide induced CD11a/CD18-dependent T cell aggregation, showing that this ligand is able to increase the avidity of CD11a/CD18 (37) . Furthermore, NK cell binding and cytotoxicity to K562 were increased by this peptide, suggesting that ICAM-2 also triggers signal transduction (37) .

In this study we investigated the effect of the ICAM-2 peptide on NK cell motility. Our results show that this peptide, but not ICAM-1 nor ICAM-3 peptides made from the homologous regions, significantly increased endogenous NK cell migration and that the migration was inhibited by anti-CD11a mAb. A comparable increase of migration was achieved when the CD11a or CD18 polypeptides were cross-linked with specific antibodies. Both the ICAM-2 peptide and antibody cross-linking increased actin polymerization and cell spreading, showing that they can induce changes in the actin-cytoskeleton.

Compounds that increase cell migration are usually soluble chemotactic or chemokinetic factors. The ICAM-2 peptide that we used in our experiments increased NK cell migration both as soluble and when substrate-bound (not shown) forms. It is well known that soluble ICAM-1 exists in human blood, and it is increased in various diseases (52, 53, 54, 55) . In addition, Makgoba (56) has recently found that soluble ICAM-2 also circulates in blood and is increased in older people. Yet the role of these soluble ICAMs is unknown, but it has been suggested that they may down-regulate inflammation (55) . However, we cannot exclude the possibility that the soluble peptide would also attach to the substrate, and lymphocytes would recognize it in solid phase-coupled form only. Such substrate-bound haptotactic or haptokinetic factors can increase cell migration (57) . Unsoluble attractants are especially necessary on endothelium where soluble factors would quickly be diluted in circulating blood (16) . Because ICAM-2 is predominantly expressed on endothelium (29) our results suggest that ICAM-2 could be involved in the activation of cell migration and extravasation.

When an attractant molecule binds to the neutrophil surface, it is rapidly internalized, and the receptor-ligand complex dissociates in the cytoplasm (58) . Free receptors recycle in 3-10 min back to the cell surface (59) , probably to the leading edge of the cell where they can bind to another attractant molecule. Higher migratory activity is detectable in a few minutes after the attractant binding (60) . In our experiments the first signs of activation of human NK cells by the peptide, i.e. clustering or spreading of the cells, were seen after 30 min to 1 h of stimulation, but only after 4 h of incubation could the migration increment be measured. Also, upon activation by cross-linking, the kinetics was about the same: 1.5-2 h after the cross-linking the first free receptors had reappeared, but only after 4 h of incubation it was seen that migration had significantly increased. Probably the increase of migration already takes place earlier, but the sensitivity of the Boyden chamber method is not sufficient for its detection.

Usually the role of chemotactic factors, besides stimulating locomotion, is to increase cell adhesion. In neutrophils, chemotactic factors cause the exposition of cytoplasmic adhesion receptor storage on cell surface. After activation, the amount of adhesion receptors can increase in a few minutes up to 10-fold (61) . In our experiments, the ICAM-2 peptide that caused increased migration of NK cells, either in a chemotactic or chemokinetic way, was itself the ligand of the adhesion receptor and did not increase the amount of CD11a/CD18 receptors on cell surface. Therefore, our results suggest that the peptide stimulates locomotion through direct signaling and up-regulated avidity of CD11a/CD18.

When migratory activity is plotted against attractant concentration, a bell-shaped curve is typical for a chemotactic factor. This is logical because at higher concentrations of a chemoattractant, near foci of inflammation, cells should slow down the migration and finally stop and adhere on the target cell (60) . We did not see any bell-shaped curve with the ICAM-2 peptide. This may be biologically meaningful since ICAM-2 is expressed by endothelium, the site that in inflammation is the starting place rather than the final goal for migrating leukocytes.

Surprisingly, 7E4 mAb did not inhibit NK cell cytotoxicity, but it inhibited migration, whereas IB4 mAb inhibited both cytotoxicity and migration (22, 23) . These results suggest that IB4 and 7E4 bind to different epitopes and that the epitope responsible for the binding to a target cell differs from the epitope which is responsible for the migration. 7E4 mAb has been shown to inhibit binding of endothelial cells to purified CD11a/CD18 (29) and aggregation of phorbol 12-myristate 13-acetate-stimulated neutrophils (41) . IB4 mAb has also been shown to abrogate the binding of various cells to endothelium and their transmigration (62, 63, 64, 65) . Both mAbs, which covered the epitope responsible for migration, stimulated migration when a secondary antibody was used as a cross-linking Ab. By cross-linking the receptors were internalized, and they reappeared at the surface of the cell as free and probably high avidity receptors. Several other mAbs, when cross-linked with secondary antibodies, have also been shown to cause both avidity increase in the CD11a/CD18 receptor and signal transduction (28, 66, 67) . The data suggest that the activation seen after cross-linking by antibodies is epitope-independent.

The region of CD11a/CD18 that binds ICAM-2 seems to be different from the region binding of IOT16 (anti-CD11a), 7E4, and IB4 (anti-CD18). The presence of ICAM-2 peptide did not inhibit the binding of the mAbs, and the binding of radioactive ICAM-2 peptide to purified CD11a/CD18 is not inhibited by 7E4 (43) . Furthermore, the different kinds of functional activities caused by the peptide and the mAbs suggest that their binding sites are different: the ICAM-2 peptide induced both migration and cytotoxicity, but the cross-linking of CD11a/CD18 with mAb induced only migration. Furthermore, we found as others have shown earlier (28) , that PI (4, 5) Pis hydrolyzed when CD11a/CD18 is cross-linked with the primary and secondary mAbs, but we found only very little PI (4, 5) Phydrolysis after activation by the ICAM-2 peptide. The tyrosine phosphorylation profile of NK cells was different after the two kinds of stimulation. These results together support the interpretation that the ICAM-2 peptide and the antibody cross-linking stimulate NK cells differently, and therefore probably affect different regions of CD11a/CD18.

Kanner et al. (66) have described that CD18 on T cells is linked to the tyrosine kinase-phosphatidylinositol pathway that stimulates tyrosine phosphorylation and activation of PLC-1 (66) . PLC-1 has a mass of 150 kDa and may correspond to the 150-kDa protein, which is observed when NK cells were activated through either CD11a/CD18 cross-linking or by the ICAM-2 peptide. In addition, we detected a phosphorylated 35-kDa protein in the ICAM-2 peptide-activated cells, but not in cells activated by cross-linking. A 35-kDa protein has previously been shown to coimmunoprecipitate with PLC-1 (68) with the ligation of TCR or the ligation of TCR and CD18. As suggested by Kanner et al. (66) , our data also suggest the possibility that the PLC-1 protein complex may be differently regulated depending on the type of triggering.

In order to examine signal transduction caused by the ICAM-2 peptide or by antibody cross-linking, we used specific protein kinase and phosphatase inhibitors. We first explored random migration and endogenous cytotoxicity. Random migration is known to be mediated mainly by CD11b (23) , and endogenous NK cell cytotoxicity by CD11a, CD11b, and CD11c/CD18 complexes (24) . Both of these activities are most probably dependent on transient and repeated changes between a high and a low avidity state of the integrin receptors. In migration the adhesion at the leading edge and the dissociation at the trailing edge of a cell is of pivotal importance, enabling a cell to crawl on substratum (69) . In cytotoxicity, cell needs to detach from a target cell after killing. Hedman and Lundgren (70) have shown that staurosporin, the serine/threonine kinase inhibitor, is able to induce a high avidity state of CD11a/CD18, and okadaic acid, the serine/threonine phosphatase inhibitor, inhibits the formation of a high avidity state of CD11a/CD18 on B cells. On the other hand, in the work of Miron et al. (71) , staurosporin decreased the fibronectin receptor avidity and okadaic acid delayed the formation of high avidity state of the fibronectin receptor on T cells. Both these inhibitors decreased random migration in our experiments. However, we have no information whether the inhibitory effect is due to changes in the avidity of integrin receptors. In our experiments okadaic acid enhanced endogenous NK cell cytotoxicity at 1 n M-1 µ M concentrations, but others (72, 73) have described with longer okadaic acid exposure times that low concentrations (<100 n M) of the drug increases the cytotoxicity of NK or T cells and high concentrations (>200 n M) decreases it. Staurosporin also blocked the ICAM-2-activated migration and cytotoxicity, but okadaic acid had no significant effect on these functions. Altogether, in our experiment the random migration and endogenous cytotoxicity of NK cells were dependent on the serine/threonine phosphorylation/dephosphorylation, but increased migration and cytotoxicity after the ICAM-2 activation were dependent on serene/threonine phosphorylation but not on dephosphorylation.

To conclude, we have demonstrated that an ICAM-2-derived peptide is a potent inducer of NK cell migration. The same peptide has earlier been shown to stimulate T cell aggregation and NK cell activity (37) . These data suggest that ICAM-2 is involved in lymphocyte activation, and thus the ICAM-2-2-integrin pathway has a function more complex than mere adhesion in lymphocyte physiology. Being abundant on endothelial and lymphohematopoietic cells, ICAM-2 may be especially relevant in the induction of migration as a result of lymphocyte contact with endothelium or other regulatory cells of the lymphoreticular system.

  
Table: The effect of protein kinase and phosphatase inhibitors on stimulated migration and cytotoxicity of NK cells

Enriched NK cells were preincubated with the inhibitors for 30 min and then activated. After 4 h the migration of CD56+/CD3- NK cells was calculated or the cytotoxicity of NK cells against K562 cells was measured. The values are expressed as percent of migration and cytotoxicity without the inhibitor. Mean ± S.E. of three to six experiments.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, P. O. Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland. Tel.: 358-0-4346525; Fax: 358-0-4346675.

The abbreviations used are: NK, natural killer; ICAM, intercellular adhesion molecule; LFA, lymphocyte function-associated; FITC, fluorescein isothiocyanate; cpm, counts/min; mAbs, monoclonal antibodies; TRITC, tetrametylrhodamine isothiocyanate; PLC, phospholipase C; TCR, T cell receptor.


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