1302 M. Nieto et al. Eur. J . Immunol. 1996.26: 1302-1307

Marta Nieto, Miguel Angel del Pozo and Francisco Sanchez-Madrid

Servicio de Inmunologia, Hospital de la Princesa, Universidad Aut6noma de Madrid, Madrid, Spain

Interleukin-15 induces adhesion receptor redistribution in T lymphocyte's

Chemotactic factors such as cytokines and chemokines direct the migration of leukocytes into inflammatory sites. Chemokines play a role regulating both the expression and adhesive properties of leukocyte integrins. We have recently described an additional function of chemokines in the induction of cell polariza- tion and adhesion receptor redistribution during the initial step of leukocyte locomotion. We herein report that interleukin (IL)-l5, a newly described cyto- kine with chemotactic properties, is able to induce uropod formation on T lym- phoblasts to which intercellular adhesion molecule (1CAM)-3, a leukocyte- restricted counter-receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, is redistributed. Other adhesion molecules, such as ICAM-1, ICAM-2, CD43 and CD44, also redistributed to the uropod, although in a lower proportion of the cells. The induction of uropod formation by IL-15 was observed on T lymphoblasts adhering to the integrin ligands fibronectin, vascu- lar cell adhesion molecule (VCAM)-1 and ICAM-1, but not to bovine serum albumin or poly-L-lysine. The effect of IL-15 was dose dependent and specifi- cally inhibited by a monoclonal antibody (mAb) against this cytokine. Blocking experiments with anti-IL-2 receptor p chain mAb showed an inhibitory effect on IL-15-mediated redistribution of ICAM-3, whereas no effect was observed in the presence of anti-IL-2 receptor a chain mAb. The uropod induced by IL-15 is enriched in many different adhesion receptors and, being well exposed to the external milieu, is likely to modulate the adhesive properties of lymphocytes.

1 Introduction

The cytokine IL-15 has recently been described to share with IL-2 the ability to induce T cell growth, among other biological properties [l, 21. Although these cytokines do not show sequence homology, modeling studies suggest that the three-dimensional structure of both molecules is similar. Accordingly, IL-15 is able to compete the binding of IL-2 to PHA-activated lymphocytes, indicating that IL-15 binds to some components of the IL-2 receptor (IL- 2R) complex [l]. In fact, it has been demonstrated that the cell binding of IL-15 requires the expression of the fi chain, but not the a chain of the IL-2R [2]. Very recently, a dis- tinct mouse IL-15 receptor that binds this cytokine with high affinity has been cloned and characterized [3].

The best sources of IL-15 mRNA seem to be monocytes and human epithelial and fibroblast cell lines. In contrast, activated peripheral blood T cells (PBT), a principal source of IL-2 and IFN-y mRNA, did not express IL-15 mRNA, nor did B lymphoblastoid cell lines [l]. In addi- tion, the expression of IL-15 mRNA has been detected by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses in several human tissues [l, 41. IL-15 is expressed in placenta and skeletal muscle, with lower levels of mRNA in heart, lung, liver and kidney. Epidermis and dermal fibroblasts also show a constitutive and inducible IL-15 mRNA expression, respectively [4].

Apart from its ability to induce T cell growth, it has been reported that IL-15 and IL-2 are chemoattractant factors for human PBT. In this regard, IL-15 is able to change lym- phocyte morphology from a spherical to a polarized shape, to stimulate invasion of collagen gels and to increase the proportion of motile cells [59].

[I 154181

Received February 11, 1996; accepted March 11, 1996.

Correspondence: Francisco Sinchez-Madrid, Servicio de Inmu- nologia, Hospital de la Princesa, Universidad Aut6noma de Ma- drid, E-28006 Madrid, Spain (Fax: +34-1-304-2496)

Abbreviations: FN: Fibronectin FN-40 and FN-80: 38-kDa and 80- kDa fragments of fibronectin ICAM: Intercellular adhesion mole- cule IL-2R: Interleukin-2 receptor LFA: Lymphocyte function- associated antigen MIP-1: Macrophage inflammatory protein-1 PBT: Peripheral blood T cells RANTES: Regulated on activation normal T ccll expressed and secreted VCAM: Vascular cell adhe- sion molecule VLA: Very late activation antigen

Key words: Adhesion molecule / Chemotactic factor / Lymphocyte polarization

The inflammatory response involves a cascade of molecu- lar events which include the adhesion of leukocytes to endothelium followed by their emigration into surround- ing tissues [6, 71. After leukocyte tethering and rolling, cells strongly adhere to the endothelium (arrest). There- after, migration into tissue is directed by locally active pro- migratory factors. Aside from the regulation of the expres- sion and affinity of adhesion receptors and chemoattrac- tion of lymphocytes by soluble factors, little is known about other factors that contribute to trigger the adhesion and to direct extravasation of inflammatory cells [7].

When lymphocytes become activated, different phenom- ena take place, such as activation of integrin receptors [6]

0014-2980/96/0606- 1302$10.00 + .25/0 0 VCH Verlagsgesellschaft rnbH, D-69451 Weinheim, 1996

Eur. J. Irnrnunol. 1996.26: 1302-1307 IL-15 induces adhesion receptor redistribution in T lymphocytes 1303

and cell polarization. We have recently reported that the interaction of T cells with endothelial cells elicited the polarization of lymphocytes, with formation of a mem- brane projection, termed uropod, where ICAM-3 and other adhesion molecules are concentrated [8, 91. Chemo- tactic and pro-inflammatory chemokines, such as RAN- TES, MCP-1, MIP-la, MIP-1P and IL-8, are also able to induce the formation of this uropod in T cells adhering to their endothelial counter-receptors or extracellular matrix proteins [8]. In addition, it has been proposed that the uro- pod may have a key role in the recruitment of lymphocytes to inflammatory foci [lo]. We describe herein the ability of IL-15 to induce uropod formation and cell adhesion recep- tor (ICAM-3, -1 -2, CD43 and CD44) redistribution in PBT. This is a novel biological activity of IL-15 which might be implicated in the regulation of leukocyte adhe- sion.

2 Materials and methods

2.1 Antibodies, cytokines and reagents

The anti-ICAM-3 HP2/19 and TP1/24, anti-CDlla TP1/40, anti-CD43 TP1/36, anti-CD44 HP2/9, anti-CD45 D3/9, anti-CD25 TP1/6 mAb have been described [ll-151. The anti-ICAM-1 MEM 111 was a generous gift of Dr. V. Horejsi (Institute of Molecular Genetics, Videnska, Czech Republic.). The anti-ICAM-2 CBR-IC2/2 was a kind gift of Dr. T. A. Springer (Harvard, Boston, MA). Recombi- nant IL-15 (specific activity 4.65 x los U/pg) derived from a simian kidney epithelial cell line and the anti-IL-15 mAb were provided by Immunex (Seattle, WA). IL-2 (1.8 x 104U/pg) was kindly provided by Eurocetus (Am- sterdam, The Netherlands). Recombinant human (rh) RANTES (specific activity 2-5 X 10l U/pg, purity > 97 70, endotoxin level < 0.1 ng/pg cytokine) was purchased from R and D Systems (Minneapolis, MN). rhMCPl (purity > 990/,) and anti-MCP1 mAb were purchased from Repro- Tech, rhTNF-a (sp. act. 5 X lo7 U/mg, purity > 95%) was provided by Genentech, San Francisco, CA). The anti-IL- 2-RP mAb was purchased from Pharmingen (San Diego, CA).

2.2 Protein substrata

Recombinant chimeric ICAM-1-Fc and VCAM-1-4Fc, consisting of the total extracellular integrin domains fused to the IgGl Fc fragment, were obtained as described [8, 161. Briefly, COS-7 cells were transiently transfected with pICAM-1-Fc or pVCAM-1-4D-Fc (ICAM-1 and VCAM- 1-4D cDNA cloned in pCDMSIgG1). After 4 days, prote- ins in the culture supernatants were precipitated with ammonium sulfate, and thereafter chimeric proteins were isolated by using protein A-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden). The tryptic 38-kDa and 80- kDa fibronectin fragments (FN40 and FNSO) were a gener- ous gift of Dr. A. Garcia-Pardo (Centro de Investigaciones Biolbgicas, Madrid, Spain). Poly-L-lysine was purchased from Sigma (St. Louis, MO). Bovine serum albumin (BSA) was purchased from Boehringer Mannheim (Man- nheim, Germany).

2.3 Cells

Human T lymphoblasts were prepared from peripheral blood mononuclear cells by treatment with 0.5% phyto- hemagglutinin (PHA) (Pharmacia) for 48 h. Cells were washed and cultured in RPMI 1640 (Flow, Irvine, Scot- land) containing 10% FCS (Flow) and 50U/ml IL-2. T lymphoblasts cultured by 7-12 days were typically used in all experiments. Before the experiment, cells were depleted of IL-2 for 24 h.

2.4 Immunofluorescence analysis

Immunofluorescence experiments were performed essentially as described [8, 91. Briefly, 2 x lo6 T lympho- blasts were incubated in flat-bottom, 24-well plates (Cos- tar Cambridge, MA) in a final volume of 500 p1 complete medium on coverslips coated with different protein sub- strata. Cytokines and chemokines at different concentra- tions were added and cells were allowed to settle in a cell incubator at 37°C in 5 % COz in air. After 30min, cells were fixed with 3.7 YO formaldehyde in PBS for 10min at room temperature and rinsed in TBS (50mM Tris-HCI pH 7.6, 150 mM NaCl, 0.1 % NaN3). To visualize different membrane adhesion molecules, cells were stained with specific mAb. After washing, cells were incubated with a 1 : 50 dilution of an FITC-labeled rabbit F(ab'), anti-mouse IgG (Pierce, Rockford, IL). Cells were observed using a Nikon Labophot-2 photomicroscope with 40,60 and 100 X oil immersion objectives. The proportion of uropod- bearing cells was calculated from ten randomly chosen fields (60 X objective) of each condition and direct count- ing of total cells (400-500) and uropod-bearing cells. Prep- arations were photographed on T-MAX 400 film (Kodak, Rochester, NY).

3 Results

We have previously described the effect of chemokines in inducing uropod formation to which several cell adhesion molecules redistributed [8]. Since it has recently been described that IL-15 is a chemotactic factor for human T lymphocytes [ 5 ] , we investigated the ability of this mole- cule to induce uropod formation and adhesion receptor redistribution. First, we analyzed the effect of IL-15 on T cell polarization and ICAM-3 redistribution. We found that when T lymphoblasts which had adhered to ICAM-1 were stimulated with IL-15, a significant fraction (45-50 YO) of them formed uropods that concentrated ICAM-3 (Fig. 1B and 2). In contrast, untreated lympho- cytes showed a significantly lower proportion of uropod- bcaring cells (<20%), and ICAM-3 tended to be uni- formely distributed along the surface of these cells (Fig. 1 A and 2).

We also examined the redistribution of adhesion molecules in addition ICAM-3 to the uropod in T cells treated with IL-15. We found that ICAM-1, ICAM-2, CD43 (Fig. 1 C-E, respectively) and CD44 (not shown) also clustered on the uropod, although in a lower proportion of cells than ICAM- 3 (data not shown). In contrast, other highly expressed molecules such as CD45, p l integrins and LFA-1 did not localize to the uropod (Fig. 1 F, and data not shown).

1304 M. Nieto et al. Eur. J. Immunol. 1996.26: 1302-1307

Figure 1. Induction of uropod forma- tion and cell adhesion receptor redis- tribution by IL-15. T lymphoblasts were allowed to adhere to ICAM-1 (A-B) or FN-80 (C-F)-coated coverslips for 30 min at 37 "C in the presence (B-F) or the absence (A) of 10ng/ml IL-15. Then, the cells were fixed and stained for ICAM-3 (A-B), [CAM-1 (C), ICAM-2 (D), CD43 (E), and LFA-1 (F) as described in Sect.2.4. The original magnification was x 600.

Next, we tested the effect of IL-15 in T lymphoblasts adher- ing to cell substrates other than ICAM-1. IL-15 was found to trigger uropod formation on thc integrin ligands VCAM- 1, FN-80 and FN-40 (Fig. 2). However, the effect of IL-15 was higher in T lymphoblasts adhered to ICAM-1, a ligand for LFA-1; and FN-80, a ligand for VLA-5, while lower

levels of uropod formation were observed on VCAM-1 and FN-40, the two VLA-4 ligands. No induction was found on poly-L-lysine or BSA (Fig. 2, and data not shown). Thus, adhesion through integrins is necessary for the triggering of the uropod, sinceTlymphocytes adhered to poly-L-lysine or BSA did not display this cellular structure.

The effect of IL-15 on uropod formation and ICAM-3 redistribution was dose dependent (Fig. 3A) . IL-15 effects were observed in the range of 0.1 to 1000ng/ml, with a maximal activity between 10-100 ng/ml. Furthermore, the effect of IL-15 was specifically blocked by a mAb against IL-15, but was not affected by mAb against MCP-1. Con- versely, the effect of MCP-1 was blocked by an antibody against MCP-1, but not by a mAb against IL-15 (fig. 3B).

Figure 2. IL-15-mediated cellular polarization during T lympho- blast adhesion to different substrates. T lymphoblasts were allowed to adhere to coverslips coated either with 20 yglml ICAM-I-Fc, 10 yglml VCAM-I-Fc. 20pg/ml FN-80, 20 pg/ml FN- 40, or 100pglml poly-L-Lysine for 30rnin at 37°C in the prescnce of 10 ng/ml IL-15 or RANTES. Then. coverslips were processed as described in Sect. 2.4. The arithmetic mean k SEM of three inde- pendent experiments is shown.

The triggering of uropod formation and IL-15 was com- pared to that induced by the chemokines RANTES and MCP-1 and the cytokine IL-2 (10 ng/ml) on T lymphoblasts adhered to FN-80 (Fig.3C). RANTES and MCP-1 induced uropod formation in a higher proportion of T cells than IL-15 and IL-2, which gave comparable effects. In addition, RANTES induced a stronger response at lower doses than IL-15 (1-lOng/ml). In contrast, TNF-a, a pro- inflammatory cytokine, was unable to trigger uropod formation (Fig. 3A) .

It has been previously described that IL-15 competes with IL-2 for binding to T lymphoblasts and that antibodies

Eur. J. Immunol. 1996.26: 1302-1307 IL-15 induces adhesion receptor redistribution in T lymphocytes 1305

Figure 3. Analysis of IL-15-mediated uropod formation. (A) Dose response of IL-15-mediated uropod formation. T lympho- blasts adhered to FN-80-coated coverslips were stimulated with different doses of the IL-15 (o), RANTES (V) or TNF-a (A) for 30min. Then, fixed cells were stained for ICAM-3 and the per- centage of uropod-bearing cells was determined as described in Sect.2.4. The arithmetic mean k SEM of three independent experiments are shown. (B) Effect of anti IL-15 mAb on the induction of uropod formation by IL-15. T lymphoblasts adhered to FN80-coated coverslips were stimulated with 10 ng/ml of IL-15 or MCP-I, in the presence of 5kg/ml anti-IL-15 or 5@ml anti- MCP-1 mAb. Then, fixed cells were stained for ICAM-3 and the percentage of uropod-bearing cells was determined as described in Sect.2. The arithmetic mean f SEM of four independent experiments are shown. (C) Uropod formation and ICAM-3 redistribution induced by IL-15, IL-2 and chemokines. T lym- phoblasts adhered to FN80-coated coverslips were stimulated with lOng/ml of each cytokine or chemokine. Then, fixed cells were stained for ICAM-3 and the percentage of uropod-bearing cells was determined as described in Sect. 2.4. The arithmetic mean k SEM of four independent experiments with four different donors is shown.

Figure 4. Prevention of IL-15-mediated uropod formation by anti-IL-2R mAb. T lymphoblasts adhered to ICAM-I-Fc-coated coverslips were first incubated for 15 min with 10 pg/ml anti-ILZR p mAb or 10Opl culture supernatant of the anti-IL-2R a TP116 mAb and then stimulated with 10ng/ml IL-15, IL-2 or RANTES. After 30min, cclls were fixed and uropod formation was quanti- fied as described in Sect. 2.4. The arithmetic mean f SEM of four independent experiments is shown.

against the P-chain of the IL-2R are able to block the biological activity of IL-15 [ 11. Furthermore, the expres- sion of the IL-2R P chain, but not IL-2R a chain, appears to be required for IL-15 cell binding [2]. To study which receptors are involved in uropod formation and adhesion receptor redistribution mediated by IL-15, we performed blocking experiments with anti-IL-2R a chain and anti-IL- 2R P chain mAb. We found that the anti-IL2R P chain mAb inhibited the effects mediated by both IL-15 and IL-2 [Fig. 41. On the other hand, the anti-IL-2R a chain mAb inhibited the effect of IL-2, but not the induction of uro- pod formation triggered by IL-15 (Fig.4). None of these mAb blocked the effect of the chemokine RANTES, which is known to bind to the chemokine receptor 1 of the seven transmembrane helix receptor family [ 171.

4 Discussion

We describe here a novel biological function mediated by the recently identified cytokine IL-15. IL-15 induces uro- pod formation and redistribution of cell adhesion recep- tors. On the uropod induced by IL-15 in PBT, several cell adhesion molecules, such as ICAM-1, -2, -3, CD43 and CD44, are concentrated. It is remarkable that these mole- cules, beside their role in cell adhesion, are involved in leukocyte activation and aggregation [9, 11, 18-20].

Recently, Wilkinson and Liew [5] reported that IL-15 stimulates locomotion of human T lymphocytes, as judged by cell shape and time-lapse film analysis and invasion of collagen gels. We describe herein that IL-15 has a notice- able effect on lymphocyte polarization and cell adhesion receptor distribution. In addition, we observed that these effects require cell adhesion to integrin ligands. We have previously found that IL-15 is not the only soluble factor that induces uropod formation and clustering of adhesion molecules, since other chemoattractant molecules exert these effects [8]. Interestingly, it has been described that IL-2 acts as a chemoattractant factor for human T lym- phoblast [5]. Accordingly, our data show that IL-2 is able to induce adhesion receptor polarization at similar con-

1306 M. Nieto et al. Eur. J . Immunol. 1996.26: 1302-1307

centrations as IL-15 (data not shown). In our previous studies on uropod formation, this activity of IL-2 was not detected, most likely due to the low dose used [S].

It is of interest that IL-2 and IL-15 havc the same capabil- ity to induce uropod formation and cell adhesion redis- tribution. In this regard, it has been found that mice in which the IL-2 gene has been disrupted do not exhibit the expected degree of T cell function impairment [21, 221. In fact, these mice are able to develop inflammatory T cell- mediated responses and to reject islet cell allografts [23]; these observations suggest that the functions of IL-2 can be replaced in vivo by another cytokine, most likely IL-15. On the other hand, the functional relevance of the che- moattractant activity of both IL-2 and IL-15 has not been determined, but it is very likely that this function has an important role in some inflammatory phenomena, as well as in the cognate interaction between T lymphocytes and antigen-presenting cells. Interestingly, a recent report indi- cates that ultraviolet B light is able to induce IL-15 secrc- tion by both keratinocytes and epidermal fibroblasts [4]. Since ultraviolet B radiation can act as an immunoregula- tory and pro-inflammatory stimulus, it is conceivable that IL-15 is involved in these phenomena. It is also possible that the chemoattractant activity of IL-15 has an additive or synergistic effect with other cytokines or chemokines.

The effects of both IL-15 and 1L-2 uropod formation and adhesion receptor redistribution were blocked by mAb against the IL-2R fi chain. In contrast, the by IL-2R a chain mAb that was able to inhibit the effects mediated by IL-2, did not affect IL-15-mediated uropod formation and adhesion receptor redistribution. These data are in accor- dance with previous works which showed that anti-IL-2R fi mAb inhibits the cell binding of IL-15, as well as its effect on induction of T cell polarization [2, 51; in addition, our findings are in agreement with the requirement of the cel- lular expression of both the fi and y subunits of IL-2R for IL-15 binding [2]. Indeed, the recent cloning and charac- terization of the mouse IL-15R a chain showed that it is structurally related to IL-2R a [3]. The mouse IL-15R a chain binds IL-15 with high affinity, but requires fi chain and y chain of the IL-2R expression to induce T cell growth [31.

Like IL-15, other chemotactic factors are able to trigger cell adhesion receptor clustering into the uropod structure. It is possible that the induction of the uropod by IL-15, which is synthesized by nonlymphoid cells such as mono- nuclear phagocytes [l], could be important at sites where other cytokines or chemotactic molecules are not present. It is also possible, as stated above, that its activity syner- gizes with other chemokines at sites of lymphocyte recruit- ment.

Lymphocyte recruitment is a key step in the inflammatory process, which is closely regulated by many factors. It is very feasible that the uropod may have a role in this pro- cess, mainly because it concentrates different leukocyte adhesion molecules on an exposed projection of the cell. In addition, we have previously described that uropod formation is induced when T cells adhere to endothelial cells; furthermore, leukocyte intercellular contacts have been shown to occur through the uropod [lo, 121. Thus, it is conceivable that lymphocytes adhered to the endothe-

lium at an inflammatory site form uropods, thereby facili- tating the recruitment and activation of circulating leuko- cytes and acting as part of an amplification mechanism of the inflammatory cascade. The cytokines and chemokines released during the interaction between T cells and endo- thelium would act as chemoattractants, leading to the recruitment of more T cells, Thus, IL-15 is a chemoattrac- tant that may contribute to this interaction in which other cytokines and chemokines could also be involved. Although it is not known whether endothelial cells pro- duce IL-15, other nonlymphoid cells have been shown to secrete it [ l] . Further studies on the involvement of IL-15 in thc proposed mechanism are currently being carried out in our laboratory.

We are grateful to Dr. D. Cosman of Immunex Corporation, Seat- tle. WA, for the generous gifts of IL-15 und unri-IL-IS mAb. We thunk Dr. R . Gonziilez-Ammo and Dr. M . Goniez for critical conz- ments on the rnunuscript. This work was supported by grants from SAF 9610039 from Ministerio rle Educucion y Ciericia and FISS- INSALUD 95/0212 (to FS-M).

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