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Prepublished online as a Blood First Edition Paper on June 28, 2002; DOI 10.1182/blood-2002-03-0841.
CHEMOKINES
From the Department of Immunology and Oncology,
Centro Nacional de Biotecnología/Consejo Superior de
Investigaciones Cientificas (CSIC), Madrid, Spain;
Laboratoire de Physiologie, Faculté de Médecine de
Rangueil, Toulouse, France; and Unité de Microscopie
Intravitale, Laboratoire de Biologie Vasculaire, Toulouse,
France.
Homing of blood-borne lymphocytes to peripheral lymph nodes (PLNs)
is a multistep process dependent on the sequential engagement of
L-selectin, which mediates lymphocyte rolling along the luminal surface
of high endothelial venules (HEVs), followed by activation of
lymphocyte integrins and transmigration through HEVs. Within lymphoid
tissue, B and T lymphocytes then migrate toward specific microenvironments such as B-cell follicles and the paracortex, respectively. The lymphocyte-expressed chemokine receptor CCR7 is
playing an important role during this process, as its HEV-presented ligands CCL19 and CCL21 can trigger rapid integrin activation under
flow in addition to inducing a chemotactic response, which may
participate in transmigration and/or interstitial migration. Here, we
report that Tyrphostin (Tyr) AG490, a pharmacological inhibitor of
Janus family tyrosine kinases (Jaks), blocked the chemotactic
response of primary mouse lymphocytes to CCL19 and CCL21 in a
dose-dependent manner. Furthermore, Tyr AG490 inhibited rapid
CCL21-mediated up-regulation of Lymphocytes continually recirculate through the
body.1-3 They leave the blood circulation preferentially
in secondary lymphoid organs (SLOs) such as spleen, peripheral and
mesenteric lymph nodes (PLNs and MLNs, respectively), and Peyer patches
(PPs), and return to the blood via efferent lymphatic vessels and the thoracic duct.1-3 Lymphocyte recirculation is considered
to be essential for maintaining an effective immune system: in T- and B-cell areas of SLOs, antigen-presenting cells (APCs) and lymphocytes are in close physical contact, which is thought to allow for extensive "screening" of major histocompatibility complex molecules and initiation of specific immune responses.2-4
In recent years, the molecular mechanisms allowing lymphocytes to enter
SLOs have been thoroughly investigated. In PLNs, these studies have
established a multistep adhesion cascade.2 In a first
step, L-selectin on blood-borne lymphocytes binds to peripheral node
addressin (PNAd) expressed on specialized postcapillary venules, the
high endothelial venules (HEV).2,3 L-selectin engagement functions as a "molecular brake," allowing interacting cells to slowly roll along the surface of HEVs in the direction of the blood
flow; however, it does not confer firm adhesion.5
Subsequently, the chemokines CCL19 and CCL21 presented on the
luminal surface of HEVs6-8 bind and activate the chemokine
receptor CCR7 on rolling lymphocytes.8,9 This event
triggers a signaling cascade resulting in the rapid (< 1 second to a
few seconds) activation of adhesion receptors of the integrin family
such as leukocyte function-associated antigen-1
(LFA-1).5,9-11 A similar process takes place in PP HEVs, where Despite recent advances, the knowledge about intracellular signaling
pathways associated with lymphocyte homing is still incomplete, especially molecular events elicited by CCR7, leading to rapid integrin
activation and interstitial migration. The current paradigm for
chemokine receptor signaling involves activation of G Some of the proposed signaling pathways have been confirmed for CCR7,
such as blocking of Ca++-flux, chemotaxis, rapid integrin
activation, and lymphocyte homing by pertussis toxin (PTX), a
G To further characterize intracellular signaling molecules involved in
physiological lymphocyte recirculation, we tested a number of
pharmacological inhibitors for their effect on lymphocyte migration
toward CCR7 ligands. Here, we report that the Jak inhibitor Tyr AG490
blocks chemotaxis of primary mouse lymphocytes to CCL19 and CCL21, as
well as to CXCL12. In addition, we analyze the effect of Tyr AG490 on
rapid integrin activation in vitro on a reconstituted endothelial
surface as well as in an in vivo model of the PLN microcirculation.
Finally, we provide biochemical evidence for rapid, G Antibodies and reagents
Anti-Jak2 rabbit polyclonal IgG was obtained from Upstate
Biotechnology (Lake Placid, NY), and agarose-coupled anti-PTyr (PY-20) was from Transduction Laboratories (Lexington, KY). Pertussis toxin
(PTX), wortmannin (Wn), Ly294002 (Ly), Tyrphostin (Tyr) AG490, Tyr AG9,
and phorbol-12-myristate-13-acetate (PMA) were purchased from
Calbiochem (La Jolla, CA). Cholera toxin (CTX) was from Sigma (St
Louis, MO). Murine CCL19, human ICAM-2/Fc fusion protein, and VCAM-1
were obtained from R&D Systems (Minneapolis, MN). Murine CCL21 and
human CXCL12 were from Peprotech (London, United Kingdom). The murine
pre-B-cell line L1-2 transfected with mCCR7
(L1-2mCCR7)8 was generously provided by Dr
Martin Dorf, Harvard Medical School. L1-2mCCR7 was
maintained in complete medium-RPMI (CM-R; RPMI/10% fetal calf serum
[FCS]/standard supplements).
Isolation of human PNAd
Inhibitor treatment of mouse lymphocytes Peripheral and mesenteric lymph nodes (LNs) from male or female BALB/c mice (5-7 weeks old) were isolated and passed through a cell strainer (Becton Dickinson, Franklin Lakes, NJ) in prewarmed labeling medium (LM; Dulbecco modified Eagle medium/10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.5/5 mM EDTA [ethylenediaminetetraacetic acid]). After resuspension in flow chamber/chemotaxis medium (FCM; RPMI/1% FCS/10 mM HEPES, pH 7.5) to 1 × 106 cells/mL, mouse lymphocytes were pretreated with inhibitors or dimethyl sulfoxide (DMSO) (1 µL/mL) for 2 hours at 37°C, 5% CO2. The final concentrations of the inhibitors were 0.1 µg/mL PTX, 0.4 µg/mL CTX, 0.5 µM Wn, 20 µM Ly, and 100 µM Tyr AG490, unless stated otherwise. Inhibitors dissolved in DMSO (Wn, Ly, and Tyr AG490) were added at a 1:1000 dilution to cells. Under these conditions, inhibitor or DMSO treatment did not affect viability as assessed by forward/side scatter characteristics and trypan blue exclusion (not shown).For some experiments, lymphocytes (5 × 106
cells/mL) were stimulated for 24 hours with Chemotaxis assays Chemotaxis assays with control- or inhibitor-treated lymphocytes (5 × 106 cells/mL) were carried out according to the manufacturer's instructions (Transwell 5-µm pore size, CoStar, Cambridge, MA). After 2 hours at 37°C, 5% CO2, migrated cells were counted on a flow cytometer (Coulter, Miami, FL) for 30 seconds and compared with a precalibrated bead standard population (Sigma). From this, the percentage of migrated cells was calculated.Static adhesion assays Static adhesion assays were carried out as described with slight modifications.9 Briefly, 12-well slides were coated with 20 µL/well of VCAM-1 (10 µg/mL in phosphate buffered saline [PBS]) or ICAM-2/Fc (5 µg/mL) overnight in a humidified chamber at 4°C. After blocking the wells with 20 µL FCS for 15 minutes at 37°C, 1 × 105 cells in 16 µL were added to each well (in duplicates) and allowed to settle for 15 minutes at 37°C, 5% CO2. To maintain cells at a physiological temperature during the experiment, the slides were placed on a prewarmed (37°C) metal block. CCL21 (4 µL/well) was added to the 12 o'clock position of each well, except the 0 minute time point, to a final concentration of 1 µM. Under these conditions, cells adhere adjacent to the site of chemokine addition.9 Adhesion is specific, as it requires precoated VCAM-1 or ICAM-2 and can be blocked by anti-integrin mAbs and PTX (not shown). In some experiments, PMA (final concentration 0.1 µg/mL) was added instead of CCL21. Unbound cells were washed off by dipping the slide twice (once from each direction) in ice-cold Hanks balanced salt solution (HBSS)/10 mM HEPES, pH 7.5, followed by fixation for 1 hour in ice-cold HBSS/10 mM HEPES, pH 7.5/1.5% glutaraldehyde. Adherent cells were counted using National Institutes of Health (Bethesda, MD) image software 1.62.Flow chamber assays Bacteriological petri dishes were coated with a mixture of PNAd (1:20 dilution) and VCAM-1 (10 µg/mL) with or without CCL21 (2 µM) in a total volume of 15 µL PBS. After overnight incubation at 4°C in a humidified chamber, the coated spot was washed 4 times with PBS and blocked with 200 µL FCS for 15 minutes at 37°C. The substrate-coated petri dishes were incorporated as the lower wall of a parallel flow chamber (IQUUM, Boston, MA) and mounted on an inverted microscope (Olympus, Tokyo, Japan) connected to a CCD camera (Cohu, San Diego, CA). Cells pretreated with DMSO or Tyr AG490 (1 × 106/mL) were infused for 2 minutes at 1 mL/min before the flow rate was adjusted to 1 dyne/cm2 (0.204 mL/min) and observed with a 10 × objective. During the experiment, mouse lymphocytes were kept in a 37°C water bath. Events were recorded on a VHS videocassette recorder (Sony, Madrid, Spain) for 4-5 minutes for later off-line analysis. Adherent cells were washed off with RPMI/5 mM EDTA for 3-4 minutes followed by RPMI/10 mM HEPES, pH 7.5, for 5 minutes at 1 mL per minute. Subsequently, the second cell population was filmed in the same field of view. Control- and Tyr AG490-treated mouse lymphocytes were infused in an alternating order to control for an eventual wash-out effect of CCL21 caused by prolonged perfusion during the course of the experiment.Interacting cells were analyzed off-line during 3 minutes for rolling
cells (rollers per minute). Cells already interacting when coming into
the field of view as well as newly tethering cells were included.
Rollers that became firmly adherent (stationary for Intravital microscopy of mouse subiliac LN Intravital microscopy (IVM) of mouse subiliac lymph node venules has been described in detail previously.27 Briefly, BALB/c mice (Charles River, St Germain sur l'Arbresle, France) were anesthetized by intraperitoneal injection of 5 mg/mL ketamine and 1 mg/mL xylasine (10 mL/kg) and surgically prepared under a stereomicroscope (Leica Microsystems SA, Rueil-Malmaison, France) to allow exposure of the node vessels. A catheter was inserted in the contralateral femoral artery to permit subsequent retrograde injections of fluorescent cell suspensions or Tyr AG490. The mouse was then transferred to an intravital microscope (INM 100; Leica Microsystems SA). Body temperature was maintained at 37°C using a padding heater. Lymph node vessels and fluorescent cells were observed through 10 × or 20 × water immersion objective (Leica Microsystems SA) by transillumination or epifluorescence illumination. Transilluminated and fluorescent events were visualized using a silicon-intensified target camera (Hamamatsu Photonics, Massy, France) and recorded for later off-line analysis (DSR-11 Sony, IEC-ASV, Toulouse). In some experiments, calcein-labeled Tyr AG490-treated mouse lymphocytes were injected first and their behavior inside the node microcirculation recorded. Control-treated cells were injected after a waiting period of 15 minutes to allow disappearance of recirculating cells from the previous injection. Alternatively, control cells were injected first, followed by Tyr AG490-treated lymphocytes. In these experiments, injection of Tyr AG490-treated cells was preceded by injection of 200 µL Tyr AG490 (100 µM).Lymphocyte behavior in lymph node vessels was analyzed off-line as
described.27 Briefly, the rolling fraction was determined in every visible lymph node HEV as the percentage of lymphocytes interacting with the endothelial lining over the total cell number entering the venule during an observation period. Rolling cells that
became subsequently adherent were included in the rolling fraction. The
sticking fraction was determined as percentage of rollers that became
firmly adherent in HEVs for more than 20 seconds. Only vessels
with Immunoprecipitation studies Immunoprecipitations were essentially carried out as described earlier.17 In brief, cells (3 × 107/mL freshly isolated or 2 × 107/mL CD3/CD28-activated mouse lymphocytes) were stimulated with CCL21 (100 nM final concentration) in a 37°C shaker at 130 rpm. After indicated times, cells were immediately transferred on ice, centrifuged, and lysed in lysis buffer (1% NP-40/137 mM NaCl/1 mM MgCl2/1 mM CaCl2/20 mM Tris [tris(hydroxymethyl)aminomethane]-HCl, pH 8.0/10% glycerol/protease inhibitor cocktail/100 µM orthovanadate) for 30 minutes at 4°C. Lysates (650 µg) were immunoprecipitated with anti-PTyr overnight at 4°C. Immunoprecipitates or cell lysates were separated by 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, blocked with 5% milk in Tris-buffered saline (TBS), and revealed with anti-Jak2 Ab.Statistical analysis Data were analyzed using InStat software (GraphPad Software, San Diego, CA). For flow chamber assays and IVM experiments, that is, when observing cell populations in the same field of view or identical HEVs, the paired Student t test was employed. For all other experiments, the unpaired Student t test was used. Significance was set at P < .05. Data are presented as mean ± SD unless otherwise stated.
The protein tyrosine kinase inhibitor Tyr AG490 blocks lymphocyte migration to CCL21 without interfering with CCR7 expression In an attempt to characterize signaling molecules involved in physiological lymphocyte recirculation mediated by CCR7, we treated freshly isolated mouse lymphocytes with a number of previously described inhibitors of chemokine receptor signaling and analyzed their effect on cell migration toward CCL21 and CXCL12 in a chemotaxis assay. As predicted, PTX almost completely abolished chemotaxis of mouse lymphocytes to 100 nM CCL21, whereas CTX had little to no effect (92% ± 6% versus 9% ± 6% inhibition compared with untreated mouse lymphocytes at 100 nM CCL21; mean ± SD). The PI3-kinase inhibitors Wn and Ly had a small but reproducible effect on lymphocyte migration (20% ± 4% and 30% ± 2% inhibition compared with migration to their diluent, DMSO) to CCL21 (Figure 1A). Similar results were obtained when cells migrated to 50 nM CXCL12 (Figure 1B). Interestingly, we also found a reduction of mouse lymphocyte migration to CCL21 and CXCL12 in the presence of the tyrosine kinase inhibitor Tyr AG490 (Figure 1A-B; 77% ± 7% inhibition and 67% ± 3% to CCL21 and CXCL12, respectively, at 100 µM Tyr AG490). This inhibition was observed over a wide range of CCL21 concentrations (Figure 1C). Likewise, migration to 100 nM CCL19 was blocked by Tyr AG490 (Figure 1D). Inhibition by Tyr AG490 was dose dependent, becoming apparent at 50 µM (not
shown). Tyr AG490 also reduced chemotaxis of a murine pre-B-cell line
stably transfected with mCCR7 (L-12mCCR7) to 25 nM CCL21 by
76% ± 14%, whereas Tyr AG9, a chemically related molecule that
serves as negative control for Tyr AG490, did not exhibit a blocking
effect on chemotaxis (not shown).
The inhibitory effect of Tyr AG490 for migration toward CCL19 and CCL21 was similar in CD4-, CD8-, and B220-positive mouse lymphocytes and also observed in freshly isolated human peripheral blood lymphocytes (PBLs; not shown). It is important to note that Tyr AG490, being a reversible inhibitor, had to be present at all times during the chemotaxis assay to exert an inhibitory effect; pretreatment alone was not sufficient to reduce the number of migrated cells (not shown). To exclude nonspecific effects of Tyr AG490 on lymphocytes, we
determined the levels of CCR7 and adhesion molecules by flow cytometry.
As an indirect measure for CCR7 surface expression, we employed
CCL19-Ig fusion proteins.15,25 The number of CCL19 binding
sites was not affected by Tyr AG490 treatment (Figure 1E). Likewise,
levels of L-selectin, LFA-1, and Tyr AG490 blocks rapid integrin activation under static conditions In addition to evoking a chemotactic response, CCL21 also can rapidly up-regulate integrin adhesiveness on lymphocytes.9-11,22 To test whether tyrosine kinases may be involved in this process, mouse lymphocytes pretreated with DMSO or Tyr AG490 were added to multiwell glass slides coated with the very late antigen 4 (VLA-4) 4 1 ligand VCAM-1 and exposed for 0 to 5 minutes to 1 µM CCL21 (Figure 2A). Under these conditions, maximal
adhesion is observed 1 to 3 minutes after chemokine
addition.9,22 Lymphocytes pretreated with Tyr AG490
adhered significantly less than control cells (63% ± 15%
inhibition at 2 minutes after chemokine addition). A similar result was
obtained when we compared DMSO- or Tyr AG490-treated L1-2mCCR7 cells binding to VCAM-1 (59% ± 15%
inhibition; not shown).
We found that commercially available recombinant ICAM-1 and -2 are poor ligands in static adhesion assays for freshly isolated lymphocytes (not shown); however, adhesion is increased when lymphocytes are activated for 24 hours with anti-CD3/CD28 mAbs prior to adhesion assays. Taking advantage of this, we tested LFA-1-mediated binding of activated mouse lymphocytes to recombinant ICAM-2. Tyr AG490 treatment almost completely abolished adhesion to ICAM-2 (87% ± 2% inhibition; Figure 2B). Importantly, when integrins were activated with PMA, no significant difference in adhesion to VCAM-1 was observed between control and Tyr AG490-treated cells (Figure 2C). These data suggest that Tyr AG490 blocks a signaling pathway triggered by CCL21 binding to CCR7 but does not interfere with signaling when integrins are activated by a chemokine-independent pathway. Activation of
The slightly elevated rolling flux in Tyr AG490-treated cells is most likely due to their impaired ability to undergo firm adhesion, thereby increasing the absolute number of rolling cells entering the field of view. When we compared the rollers per minute in the absence of CCL21, we found no difference between both samples (not shown), which is also consistent with the similar expression levels of L-selectin in both cell samples (Figure 1E). In situ analysis of control- and Tyr AG490-treated mouse lymphocytes in the PLN microcirculation To corroborate our in vitro findings in a more physiological setting, we investigated the in vivo role for tyrosine kinases during lymphocyte migration to secondary lymphoid organs. In preliminary experiments, we carried out homing assays with fluorescently labeled control- and Tyr AG490-pretreated cells; however, no difference was found after a 2-hour homing period (not shown). Based on the previously observed reversibility of Tyr AG490 inhibition (see "Results"), we reasoned that the lack of inhibition may be due to a wash-out effect once pretreated lymphocytes entered the blood circulation. This prompted us to choose a more direct approach to test the effect of Tyr AG490 during physiological homing by employing intravital microscopy of the PLN microcirculation.5,8,27 This allowed us to carry out a real-time analysis of lymphocyte behavior immediately after injecting the cells into the bloodstream, which we hypothesized would enable us to detect an inhibitory effect of Tyr AG490 before the inhibitor diluted out of cells. Consistent with results from flow chamber experiments, Tyr AG490 did not significantly alter the cells' ability to undergo L-selectin-mediated rolling in HEVs (61% ± 32% control versus 53% ± 32% Tyr AG490-treated cells; Figure 4A). However, the transition from rolling to firm adhesion was severely impaired in Tyr AG490-treated lymphocytes in all venules analyzed (42% ± 23% control and 11% ± 9% Tyr AG490-treated cells; Figure 4B). Of note, recirculating Tyr AG490-treated lymphocytes recovered their ability to arrest in PLN HEVs when the observation period was extended for more than ~ 20 minutes, suggesting that the blocking effect is reversible due to washing out of the inhibitor (not shown).
Rapid Jak2 phosphorylation after CCL21 binding to CCR7 Tyr AG490 has been characterized as a Jak2-specific inhibitor.28 As the above results suggested a rapid activation of Jak family members after chemokine binding to CCR7, we performed a biochemical analysis of early signaling events in mouse lymphocytes. We consistently observed a rapid phosphorylation of Jak2 after exposure of cells to CCL21, reaching a peak at 1 minute after chemokine addition, then decreasing to background levels within 3 to 5 minutes (Figure 5A). Similar results were obtained with preactivated lymphocytes (Figure 5B) and L1-2mCCR7 cells (not shown). CCL21-induced Jak2 phosphorylation was strongly reduced by pretreatment with Tyr AG490 but not by PTX (Figure 5C). These data are consistent with results previously obtained in human cells showing Gi-independent Jak activation in response to
various chemokines.17-20
The chemokine receptor CCR7 fulfills at least 2 important
functions during lymphocyte recirculation: rapid activation of
integrins and interstitial migration. Here, we show that a
pharmacological tyrosine kinase inhibitor, Tyr AG490, blocks both
processes in primary lymphocytes. In previous studies, pretreatment of
encephalitogenic T-cell lines with Tyr AG490 was shown to reduce their
in vitro adhesion to brain endothelium and purified
VCAM-1.29,30 In addition, systemic administration
of Tyr AG490 protected SJL/J mice from developing experimental
autoimmune encephalomyelitis.29,30 However, in vitro
adhesion in these studies was not induced by addition of chemokines and
measured over a relatively long time frame ( When using pharmacological inhibitors, nonspecific effects on treated cells cannot be fully excluded. However, several lines of evidence make it plausible that Tyr AG490 acts as a specific inhibitor with only limited side effects. First, it has previously been demonstrated that high doses of Tyr AG490 do not affect viability, proliferation, or activation of T lymphocytes.28-30 Consistent with this, we have not observed a decrease in viability of lymphocytes in our studies of Tyr AG490 treatment (not shown). Second, Tyr AG490 does not reduce lymphocyte adhesion when integrins are activated by a chemokine-independent mechanism, such as via PMA. Third, surface expression levels of CCR7 and adhesion molecules are not altered by Tyr AG490. Finally, Tyr AG490 has been described originally as an inhibitor of the Janus family kinase member Jak2,28 although a recent study also suggests a blocking effect on the closely related Jak3.31 Consistently, we observed rapid phosphorylation of Jak2 after exposure of primary lymphocytes to CCL21. Jak2 is best characterized for its role in the signaling of cytokine
receptors needed for maintenance and proliferation of hematopoietic
precursors, such as the erythropoietin (Epo) receptor (Epo-R),
interleukin 3-R (IL-3-R), granulocyte-macrophage colony-stimulating factor R (GM-CSF-R), thrombopoietin R (Tpo-R), IL-5-R, and interferon Of note, the kinetics of Jak2 phosphorylation that we observed were
considerably faster than those elicited by Epo-R or other cytokine
receptors.35 Jak2 phosphorylation in mouse lymphocytes peaked after 1 minute, returning to background levels within 3 minutes
of chemokine exposure. Similar results were obtained in L1-2mCCR7 cells (not shown) and are consistent with
previous reports on rapid Jak phosphorylation after chemokine
binding.17-21 This raises interesting questions about the
mechanism by which Jak2 transmits signals from the chemokine receptor.
A dimerization model has been previously suggested, similar to the
cytokine receptor pathway, based on ample experimental evidence that
some chemokine receptors can be coimmunoprecipitated as dimers or
oligomers after ligand binding.36 According to
this model, 2 chemokine receptors dimerize upon ligand binding and bind
Jak family members, which then phosphorylate the CCR. Jak activity was
found to be not only independent of, but also necessary for G In summary, we have identified a new role for Jak during CCR7-mediated lymphocyte recirculation using pharmacological tyrosine kinase inhibitors. The data presented here therefore support the concept that Janus kinases are playing a role for lymphocyte homing and chemokine receptor signaling pathways. Our study also suggests that intracellular molecules may serve as drug targets in order to inhibit harmful leukocyte extravasation, such as during autoimmune disorders. Further experiments using reconstituted mice genetically deficient in Jak2 will give a more detailed insight into the role of this protein during lymphocyte homing.
We would like to thank Catherine Mark for editorial assistance, as well as Dr Ulrich H. von Andrian (CBR, Harvard Medical School, Boston, MA) for continuous support. We would furthermore like to thank Dr Tim Springer, Dr Ulrich H. von Andrian, Dr Martin E. Dorf, and Dr Martin Lipp for reagents, and our animal facility staff for excellent service.
Submitted March 20, 2002; accepted June 17, 2002.
Prepublished online as Blood First Edition Paper, June 28, 2002; DOI 10.1182/blood-2002-03-0841.
Supported by the Spanish Council for Scientific Research (CSIC), the Pharmacia Corporation, Région Midi-Pyrénées, Association pour la Recherche sur le Cancer (ARC), Ligue Contre le Cancer, and Génopôle-Ministère de la Recherche. J.V.S. is a recipient of a Human Frontiers Long Term Fellowship.
S.F.S. and C.M. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jens V. Stein, Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autonoma (UAM) Campus de Cantoblanco, E-28049 Madrid, Spain; e-mail: jstein{at}cnb.uam.es.
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Top
Abstract
Introduction
represents
control; 
, Tyr AG490. (B) CCL21-stimulated adhesion of
lymphocytes on ICAM-2 in control- and Tyr AG490-treated lymphocytes at
0 and 2 minutes after CCL21 addition (n = 3; mean ± SD). 
represents t = 0 minutes; 
, t = 2 minutes. (C) PMA-induced
lymphocyte adhesion on VCAM-1. Adhesion was determined 6 minutes after
PMA addition. PMA-induced lymphocyte adhesion is dependent on precoated
integrin ligands (not shown) (n = 3; mean ± SD). 
PMA; 
R (IFN
/