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*
Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, and
Department of Immunology and Oncology, Centro Nacional de Biotecnología, CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been widely proposed that chemokines are the major soluble
mediators of leukocyte recruitment in inflammation and immunity (6, 7, 8).
These molecules are differentially secreted by platelets and a broad
spectrum of nucleated cells, including endothelial cells, T
lymphocytes, and monocytes (7, 9, 10). Although little is known about
the effects of chemokines on human NK cells, it has been shown that
these cytokines induce chemotaxis and chemokinesis of NK cells in
vitro. In addition, it has been reported that chemokines stimulate
Ca2+ mobilization and cytolytic granule release, promote
cytotoxic activity, and regulate the adhesiveness of NK cells (11, 12, 13, 14).
NK cells, in turn, synthesize chemotactic factors, including IL-8,
MIP-1
,3 and lymphotactin
(15, 16, 17), which may induce the migration of additional effector cells
into the target tissue. Chemokines specifically interact with receptors
that possess seven transmembrane domains and are coupled to a G protein
signaling pathway. Several of these receptors have been cloned and
classified into two groups, CCR and CXCR (6, 18), but their patterns of
expression on NK cells have not been fully elucidated.
Leukocyte migration requires cell polarization, a phenomenon that is involved in many other processes, such as cell differentiation, vectorial transport of molecules across cell layers, induction of immune response, cognate interactions between APC and T cells, and target cell recognition and killing (19, 20, 21, 22, 23, 24). Cell polarization is required for the release of NK cytolytic granules as well as for the formation of conjugates between killer cells and their target cells (25). We recently reported that chemokines and other chemotactic cytokines induce in T cells the polarization of chemokine receptors (CCR) to the cell leading edge, probably directing the cells along the chemokine gradient (26). In addition, chemokines induce the formation of a cell projection at the rear of the cell, termed the uropod, accompanied by redistribution of specific adhesion molecules such as ICAM-1, ICAM-3, CD43, and CD44 to this structure (27, 28, 29).
We have studied the polarization of NK cells induced by chemokines or during NK-target cell interaction as well as the possible role of the cellular uropod in the migration and recruitment of NK cells. We have found that the leading edge of NK cells concentrates chemokine receptors and is implicated in target interactions, while the uropod mediates homotypic NK cell interaction, which might be important for the recruitment of these cells into target tissues. We discuss the implication of these findings in relation to mechanisms relevant to the in vivo migration of NK cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mAbs anti-ICAM-3 HP2/19 (IgG2a), anti-CD11a TP1/40,
anti-CD45 D3/9 (IgG1), and anti-CD94 HP3B1 (IgG2a) have been
previously described (30, 31, 32). The anti-ICAM-1 MEM 111 was a gift
from Dr. V. Horëjsi (Institute of Molecular Genetics,
Videnska, Czech Republic), and the anti-CD16 B73.1 (IgG1) (33) was
a gift from Dr. B. Perussia. The anti-CD56 mAb K218 (IgG1) was
provided by Dr. A. Moretta (Instituto Nazionale per la Ricerca sul
Cancro e Centro Biotecnologie Avanzate, University of Genova, Genova,
Italy), and the L16 mAb was donated by Dr. C. Figdor (Nijmegen, The
Netherlands). Anti-CCR2 (CCR2-03) and anti-CCR5 (CCR5-01) mAb have
previously been described. Recombinant human (rh) RANTES, MCP-1,
MIP-1, MIP-1ß chemokines, and rhIL-15 were purchased from
PeproTech (London, U.K.), and rhTNF- (sp. act., 5 x
107 U/mg; purity, >95%) was provided by Genentech (San
Francisco, CA). The rhIL-2 was provided by Hoffmann-La Roche (Nutley,
NJ).
Protein substrates
Recombinant chimeric ICAM-1-Fc, consisting of the total extracellular domains of ICAM-1 fused to the IgG1 Fc fragment, was obtained as previously described (34). Briefly, COS-7 cells were transiently transfected with ICAM-1-Fc (ICAM-1 cDNA cloned in pCD8IgG1). After 4 days, culture supernatants were precipitated with ammonium sulfate, and chimeric proteins were isolated using protein A coupled to Sepharose (Pharmacia, Uppsala, Sweden). The tryptic 38- and 80-kDa fibronectin fragments (FN40 and FN80) were gifts from Dr. A. García-Pardo (Centro de Investigaciones Biológicas, Madrid, Spain). Collagen type I, laminin, poly-L-lysine, and fibrinogen were purchased from Sigma (St. Louis, MO). BSA was obtained from Boehringer Mannheim (Mannheim, Germany).
Cells
IL-2-cultured NK cells were obtained essentially as previously described (32). In brief, PBL were cultured with irradiated (5 Gy) RPMI 8866 lymphoblastoid cells for 6 to 9 days in RPMI 1640 supplemented with 10% FCS (complete medium), followed by a negative selection step using an anti-CD3 mAb plus rabbit complement (Behring, Marburg, Germany). The CD3- cells (<5% CD3+) were cultured with 50 IU/ml of rhIL2 until use. Fresh NK cell-enriched populations were obtained from PBL by removal of adherent cells on plastic petri dishes, followed by passage through a nylon wool column, and depletion of T cells with an anti-CD3 mAb plus rabbit complement (32). These cell populations are hereafter referred to as NK cells. After each purification process, the resulting population was characterized by flow cytometric analysis. We routinely obtained a cell population with percentages of CD56+ and CD16+ cells >95% and with <5% of CD3+, CD19+, or CD14+ contaminating cells. For polarization studies, NK cells were cultured in the absence of exogenous IL-2 for 12 h before the experiment. Erythroleukemic K562 target cells were cultured in complete medium.
Flow cytometric analysis
Cells were saturated with 
-globulin, incubated with the
appropriate mAb for 30 min at 4°C, then washed twice and incubated
with a 1/50 dilution of FITC-labeled rabbit F(ab')2
anti-mouse IgG (Pierce, Rockford, IL) for 20 min at 4°C.
Double-staining experiments on unfractionated PBMC were performed using
phycoerythrin-labeled anti-CD56 mAb after incubation with 10%
mouse serum. Flow cytometric analysis was performed using a FACScan
cytofluorometer (Becton Dickinson, Mountain View, CA).
Immunofluorescence analysis
The induction and detection of the uropod on NK cells were performed essentially as previously described (28). Briefly, 1 x 106 NK cells were incubated on coverslips coated with various protein substrates in flat-bottom 24-well plates (Costar, Cambridge, MA) in a final volume of 500 ml of complete medium. Chemokines and other cytokines were added at different concentrations, and cells were allowed to settle for 30 min at 37°C in a 5% CO2 atmosphere. Cells were then fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, rinsed in TBS (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% NaN3), and incubated with specific mAb. After washing, cells were stained with a 1/50 dilution of an FITC-labeled rabbit F(ab')2 anti-mouse IgG (Pierce) and analyzed using a Nikon Labophot-2 photomicroscope (Nikon, Melville, NY) with x40, x60, and x100 oil immersion objectives. The proportion of uropod-bearing cells was calculated by direct counting of total cells (n = 400–500) and uropod-bearing cells in 10 random fields (x60 objective) for each condition. Preparations were photographed on Ektachrome 400 film (Eastman Kodak, Rochester, NY).
For immunofluorescence studies of NK-K562 cell conjugates, 105 K562 cells and 2 x 105 NK cells were mixed and incubated in Ca2+-free complete medium (ICN, Costa Mesa, CA), to avoid cytolysis, at 37°C in a 5% CO2 atmosphere for 20 min in 96-well plates (Costar). Cells were then fixed with 3.7% formaldehyde, rinsed in Tris-buffered saline (TBS), stained with mAb, washed twice with TBS, and sequentially rinsed and incubated for 15 min with a biotinylated anti-mouse IgG mAb (1/300 dilution), a fluorescein-labeled avidin D (1/1000), a biotinylated anti-avidin D (1/100), and a fluorescein-labeled avidin D (1/500; all from Vector, Burlingame, CA). CCR2 and CCR5 were stained with the appropriate mAb and then incubated with a 1/50 dilution of an FITC-labeled rabbit F(ab')2 anti-mouse IgG (Pierce). Cells were observed and photographed using a Nikon Labophot-2 photomicroscope with x40 and x60 oil immersion objectives.
Time-lapse videomicroscopy
Videomicroscopy analysis was performed as previously described (35) using a Nikon Diaphot 300 inverted microscope equipped with a black and white video camera (Sony SSC-M350CE) coupled to a time-lapse videocassette recorder (Sony SVT-5000P) and a video monitor (Sony PVM-1453 MD). NK cells were allowed to attach for 30 min at 37°C to 10-mm plastic petri dishes (Costar) previously coated with ICAM-1 (10 µg/ml), in the presence of different chemokines or the polarization-inducing anti-ICAM-3 mAb HP2/19. A second cohort of unstimulated NK cells was then added, and cell-cell interactions were filmed for 1 h using a x20 phase contrast objective. When indicated, cells were pretreated for 15 min at 37°C with different blocking mAb. Images were acquired every 30 s, and sequential frames were photographed. Under each experimental condition, the number of attached cells from the first cohort that were moving on the substrate (phase-dark cells) was counted as well as the number of cells from the second cohort (phase-bright cells) that were interacting with the uropod of adhered cells. The recruitment index was expressed as the number of cells of the second cohort being captured/number of the cells of the first layer that adhered to the substrate.
Chemotaxis transwell assays
Chemotaxis assays were performed in triplicate in transwell cell culture chambers (Costar). These chambers contain an upper and a lower well separated by a tissue culture-treated polycarbonate membrane (polyvinylpyrrolidone free), 6.5 mm in diameter, 10 mm thick, and with a pore size of 5 µm. K562 target cells (5 x 105), NK cells (5 x 105), or a mixture of both cells were preincubated in a final volume of 0.6 ml of complete medium in the lower chamber for 4 h. Then, 106 51Cr-labeled NK cells were added to the upper chamber. After 2-h incubation at 37°C in 5% CO2, the number of cells that migrated to the lower chamber was calculated relative to the total input of 51Cr-labeled NK cells. Specific migration was calculated after subtracting spontaneous migration.
Chemokine quantification
K562 target cells (5 x 105), NK cells (5
x 105), or a mixture of both cells were incubated in a
final volume of 1 ml of complete medium for 4 h at 37°C in 5%
CO2. Cells were then centrifuged, and the chemokines
present in the supernatant were quantified. Human chemokines MCP-1 and
RANTES were measured using the Cytoscreen immunoassay kit
(BioSource, Camarillo, CA), and human MIP-1 and MIP-1ß were
determined using the Quantikine immunoassay kit (R&D Systems,
Minneapolis, MN), following the manufacturer’s instructions.
Cytotoxicity assays
NK cell cytotoxicity was tested against the K562 cell line in a 4-h 51Cr release assay at different E:T cell ratios, and specific lysis was calculated as previously described (32). Data are expressed as the arithmetic mean of triplicate determinations. In each case, spontaneous release was always <10% of the maximum lysis.
Statistical analysis
Significant values were determined using one-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines are produced by different cell types, including NK
cells (15, 16, 17). We analyzed whether NK cells, alone or mixed with
target cells, release soluble factors that attract additional NK cells.
Using a chemotaxis transwell assay, we found that cocultures of NK
cells with K562 target cells induced a noticeable migration of
additional NK cells, whereas K562 or NK cells alone had a weak effect
(Fig. 1
A). Measurement of
chemokines in these supernatants also revealed that NK cells produced
MIP-1, RANTES, and MIP-1ß, but not MCP-1, whereas only trace
amounts of these chemokines were found in K562 cell supernatants.
Interestingly, the secretion of these chemokines, probably by NK cells,
was dramatically increased upon coculture of NK effectors with K562
target cells (Fig. 1B). These results indicate that the
interaction of NK cells with target cells induces the release of
physiologically relevant amounts of chemokines.
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Membrane expression of CCR2, a receptor for MCP-1, MCP-3, and
MCP-4 (36), and CCR5, which specifically binds RANTES, MIP-1, and
MIP-1ß (37), was barely detectable on freshly isolated NK cells (Fig. 2aA). In contrast, CCR2 and
CCR5 expressions were up-regulated in a significant proportion of
IL-2-activated NK cells (Fig. 2aB). IL-15, which has been
reported to be a key cytokine in the development of NK cells (38, 39),
showed a comparable effect on the induction of expression of these two
chemokine receptors (Fig. 2aC). These results are
consistent with studies of the up-regulated CCR2 mRNA expression in NK
cells (40).
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and MIP-1ß induced
polarization of both CCR2 and CCR5 to the leading edge of
IL-2-activated NK cells. Neither the proinflammatory cytokine TNF-
nor the activating molecule PMA induced CCR polarization. Two integrin
ligands, FN80 and ICAM-1, supported CCR redistribution, which is
consistent with our previous data indicating that receptor polarization
requires adhesion through integrins (Fig. 2
, b andc). Chemokines also triggered the formation of the uropod and
ICAM-3 redistribution on IL-2-activated NK cells in a dose-dependent
manner (Fig. 3, a–c).
Although MIP-1 and MIP-1ß induced the uropod in a lower proportion
of cells than did RANTES and MCP-1, these chemokines showed a
significant effect at lower concentrations (0.01–1 ng/ml; Fig. 3b). IL-15, known to be a chemotactic factor, also induced
the redistribution of ICAM-3 to the uropod of NK cells and the
chemokine receptors to the leading edge (Fig. 3c and data
not shown) (26, 27, 29). Redistribution of CCR and adhesion receptors
hence correlated with the acquisition of the migratory morphology and
may guide cell migration and cell-cell interactions during NK cell
effector function.
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We next studied the role of the uropod and the leading edge in NK
cell motility, and the formation of effector-target cell conjugates by
time-lapse microscopy. IL-2-activated NK cells migrated onto
FN80-coated surfaces; they show a polarized shape, with the uropod at
the rear of the cell and the leading edge at the opposite pole. Motile
NK cells contacted the K562 target cells through the leading edge,
maintaining their polarized shape (Fig. 4a). Contacts through the
uropod were rarely observed, indicating that the existence of two
morphologic poles correlates with different functional domains.
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a). In these immunofluorescence
studies, NK cells were clearly distinguished from K562 cells based on
their polarized cell shape, lower refringence, and smaller size. CCR2
and CCR5 were localized at the area of target-effector cell-cell
contact (Fig. 4b, A–E). In contrast, ICAM-3 and
ICAM-1 were concentrated in the uropod, at the opposite side of the
cell-cell contact area (Fig. 5a, A–D). Since
K562 cells express ICAM-1, but not ICAM-2, ICAM-3, or VCAM-1, it is
likely that NK cell polarization as well as binding to K562 target
cells are mediated through the LFA-1/ICAM-1 adhesion pathway (41).
Although LFA-1 is evenly distributed, it shows a reinforced location at
the cell-cell contact areas (Fig. 5a, insets in E
and F). This was further confirmed using the L16 mAb,
which reacts specifically with an activation-dependent epitope on the
LFA-1 -chain. This Ab stained the cell-cell contact areas, but not
the uropod region (Fig. 5a, E and F).
In contrast, none of the NK cell-specific receptors, such as CD56, CD16
(FcRIII), or CD94, accumulated at the uropod; but they were evenly
distributed throughout the cell membrane (Fig. 5b,A–F). These results show that upon target cell conjugate
formation, NK cells appear to maintain a polarized shape, implying the
formation of specialized functional domains on the cell membrane.
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The fact that the uropod concentrates several
adhesion molecules in a highly exposed region of the cell prompted us
to study the role of this structure in homotypic adhesion and NK cell
recruitment. We induced cell polarization and adhesion receptor
redistribution in a layer of NK cells adhered to ICAM-1-coated plates.
Unstimulated NK cells were then added, and cell-cell interactions were
videorecorded. Following treatment with RANTES (Fig. 6), the NK cells from the first layer
migrate onto the substrate (phase-dark cells), contact unstimulated NK
cells (phase-bright cells) through the uropod, and capture and
transport them, thus moving together. These captured cells were tightly
attached through the uropod, and the cell transport phenomenon was long
lasting, persisting for at least several minutes. Quantitative analysis
of the chemokine-induced NK cell recruitment showed that MCP-1 and
RANTES clearly enhanced this phenomenon, whereas MIP-1 and MIP-1ß
had weaker activity, and TNF- had no significant effect (Fig. 7A). The strongest response
was induced by the polarization-inducing anti-ICAM-3 mAb HP2/19
(Fig. 7). The effects of MCP-1 and the anti-ICAM-3 HP2/19 mAb on
cell recruitment were specifically inhibited with the anti-ICAM-3
blocking mAb 140.11, but not with the control anti-CD45 D3/9 (Fig. 7, B and C). These results show the involvement
of chemokines in NK cell recruitment and the dependence of this
phenomenon on ICAM-3.
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). A second cohort of
nonstimulated NK cells was then added, and after 30 min, K562 cells
were added, and NK cell migration was filmed. As described above,
uropod-bearing NK cells tightly trapped NK cells from the second
cohort, which were guided by the polarized migrating NK cells to
contact the K562 cell (Fig. 8). This finding suggests that during the
cytotoxic response, adhesion receptor redistribution to the uropod may
exert an amplifying effect on cell conjugate formation by increasing
the number of NK cells available to interact with target cells.
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We have reported that disruption of the myosin motor by specific
drug inhibitors prevents cell polarization and redistribution of
adhesion receptors to the cell uropod without affecting cell adhesion
(42, 43). To further analyze the role of cell polarization and adhesion
receptor redistribution in the cytotoxic activity of NK cells, we
investigated the effects of the myosin-disrupting agent butanedione
monoxime in effector-target conjugate formation as well as in NK
cell-mediated lysis of K562 target cells. Butanedione monoxime, which
inhibited NK cell polarization and ICAM-3 redistribution to the cell
uropod (Fig. 9aB), prevented
conjugate formation (Fig. 9a, F and G)
and blocked NK cell-mediated cytotoxicity (Fig. 9b). In
contrast, nocodazole, which disassembles the microtubule network but
did not inhibit uropod formation and cell polarization (Fig. 9aC) (44), did not alter the formation of cell conjugates
(Fig. 9a, H and I). NK cell
cytotoxicity mediated by cells with disassembled microtubules was only
weakly influenced, in accordance with a previous report (Fig. 9b) (45). These results suggest that chemokines may affect
NK cell-mediated target cell conjugate formation and cytotoxicity by
regulating receptor redistribution and cell polarization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell polarization plays an important role in the immune response as well as in other biologic processes (21, 22, 23, 24, 27). For instance, T cells recognize and bind APCs through their leading edge (21, 23). Using time-lapse videomicroscopy, we observed a similar behavior for NK cells when they interact with the K562 target cells; NK cells contacted the target cells through the advancing front, a region in which CCR2 and CCR5 are concentrated, whereas the effector-target cell interactions through the uropod were rarely observed. We also found that during the formation of effector-target cell conjugates, ICAM-1 and ICAM-3 adhesion molecules are concentrated in the uropod of NK cells, which is located at the most distal point from the area of NK-target cell contact. The redistribution of adhesion molecules and CCR to the opposite poles of NK further supports the idea that cell polarization arises in two specialized regions on the cell membrane, which make feasible several key phenomena, such as cell migration and intercellular adhesion. The functional relevance of cell polarization is made evident by the fact that both CCR and adhesion receptor redistribution are maintained not only during NK cell migration, but also when they are bound to their targets. Furthermore, preventing cell polarization and adhesion receptor redistribution blocked the formation of effector-target cell conjugates and NK cell-mediated cytotoxicity, thus demonstrating the functional importance of these phenomena.
The mechanisms by which NK cells promote cytotoxic activity in specific tissues remains poorly understood. We propose the existence of a cooperative mechanism involved in NK cell migration. On the one hand, by inducing cell polarization, chemokines and cytokines play important roles in the development of the NK cell cytotoxic response. Cell polarization determines the formation of different functional domains in the NK cell. The leading edge, which concentrates the CCR, is involved in target cell adhesion, granule release during cytotoxic phenomena (25), and probably directing the cell during migration, whereas the uropod, which accumulates ICAM, is involved in leukocyte recruitment. In fact, uropod-mediated cell-cell contacts appear to recruit additional NK cells to the vicinity of the target cells, which may increase contact frequency between NK and target cells, favoring activation of the recruited cells. This concurs with our earlier studies of T lymphocytes, in which ICAM redistribution to the uropod is a cooperative mechanism in lymphocyte recruitment, acting as an amplification system during both cell extravasation and migration toward inflammatory foci (28, 35). On the other hand, the molecules inducing NK cell polarity appear to be chemokines produced by activated NK cells. A role for chemokines in the activation of NK cell-mediated cytotoxicity has also been previously reported (12, 13). In agreement with our findings, the authors found that the enhancement of NK cytotoxicity mediated by chemokines was dependent on conjugate formation (12). We have found that the formation of NK-target cell conjugates induces the release of chemokines, which are responsible for NK cell migration, a finding that further substantiates our hypothesis of a cooperative mechanism of NK cell migration to the target. Some of these events, including cell polarization and the induction of cytotoxic activity, can be blocked by ADP ribosylation of the GTP binding protein RhoA, indicating the key role of this chemokine receptor-associated signaling pathway (49). In summary, these observations are consistent with the view of the NK cell as a sensitive immune cell, responsive to external cues and able to alter its shape in response to diverse stimuli.
As occurs in vitro, it is also conceivable that the in vivo interaction of NK with target cells may induce the release of soluble factors, including chemokines probably produced by effector cells, which can trigger NK cell polarization, chemotaxis, and recruitment of additional NK cells to the tissue. It is thus entirely feasible that the in vivo recruitment of NK cells triggered by chemokines and cytokines at sites of immune activation has an important amplifying effect in the elimination of tumor cells and parasites. The induction of NK cytotoxicity by IL-15 secreted during human herpes virus infection may be an example of such a mechanism (47). IL-15 acts as a chemotactic factor that induces cell polarization (27) and adhesion receptor distribution (29) and regulates the migration of lymphocytes at inflammation sites in diseases such as rheumatoid arthritis (50). Our data suggest that chemokines have a similar in vivo role in NK cell-mediated phenomena.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Francisco Sánchez-Madrid, Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Diego de León 62, E-28006, Madrid, Spain. E-mail address:
3 Abbreviations used in this paper: MIP-1, macrophage inflammatory protein-1; rh, recombinant human; MCP, monocyte chemoattractant protein; FN, fibronectin.
Received for publication February 23, 1998. Accepted for publication June 1, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß chemokines induce NK cell migration and enhance NK-mediated cytolysis. J. Immunol. 155:3877.[Abstract]
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R (CD16) interaction with ligand induces Ca2+ mobilization and phosphoinositide turnover in human natural killer cells: role of Ca2+ in FcR (CD16)-induced transcription and expression of lymphokine genes. J. Exp. Med. 169:549., and MIP-1ß. J. Biol. Chem. 271:17161. production in rheumatoid arthritis. Nat. Med. 3:189.[Medline]
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E. M. Schneider, I. Lorenz, M. Muller-Rosenberger, G. Steinbach, M. Kron, and G. E. Janka-Schaub Hemophagocytic lymphohistiocytosis is associated with deficiencies of cellular cytolysis but normal expression of transcripts relevant to killer-cell-induced apoptosis Blood, September 26, 2002; 100(8): 2891 - 2898. [Abstract] [Full Text] [PDF]
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F. Marti, E. Bertran, M. Llucia, E. Villen, M. Peiro, J. Garcia, and F. Rueda Platelet factor 4 induces human natural killer cells to synthesize and release interleukin-8 J. Leukoc. Biol., September 1, 2002; 72(3): 590 - 597. [Abstract] [Full Text] [PDF]
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C. Dominguez-Jimenez, D. Sancho, M. Nieto, M. C. Montoya, O. Barreiro, F. Sanchez-Madrid, and R. Gonzalez-Amaro Effect of pentoxifylline on polarization and migration of human leukocytes J. Leukoc. Biol., April 1, 2002; 71(4): 588 - 596. [Abstract] [Full Text] [PDF]
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M. J. Robertson Role of chemokines in the biology of natural killer cells J. Leukoc. Biol., February 1, 2002; 71(2): 173 - 183. [Abstract] [Full Text] [PDF]
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S. Chantakru, C. Miller, L. E. Roach, W. A. Kuziel, N. Maeda, W.-C. Wang, S. S. Evans, and B. A. Croy Contributions from Self-Renewal and Trafficking to the Uterine NK Cell Population of Early Pregnancy J. Immunol., January 1, 2002; 168(1): 22 - 28. [Abstract] [Full Text] [PDF]
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J. J. Campbell, S. Qin, D. Unutmaz, D. Soler, K. E. Murphy, M. R. Hodge, L. Wu, and E. C. Butcher Unique Subpopulations of CD56+ NK and NK-T Peripheral Blood Lymphocytes Identified by Chemokine Receptor Expression Repertoire J. Immunol., June 1, 2001; 166(11): 6477 - 6482. [Abstract] [Full Text] [PDF]
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K. Kito, K. Morishita, and K. Nishida MCP-1 receptor binding affinity is up-regulated by pre-stimulation with MCP-1 in an actin polymerization-dependent manner J. Leukoc. Biol., April 1, 2001; 69(4): 666 - 674. [Abstract] [Full Text]
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 J. Trotter, C. Klein, and E.-M. Kramer GPI-Anchored Proteins and Glycosphingolipid-Rich Rafts: Platforms for Adhesion and Signaling Neuroscientist, August 1, 2000; 6(4): 271 - 284. [Abstract] [PDF]
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 D. Sancho, M. Nieto, M. Llano, J. L. Rodriguez-Fernandez, R. Tejedor, S. Avraham, C. Cabanas, M. Lopez-Botet, and F. Sanchez-Madrid The Tyrosine Kinase Pyk-2/Raftk Regulates Natural Killer (Nk) Cell Cytotoxic Response, and Is Translocated and Activated upon Specific Target Cell Recognition and Killing J. Cell Biol., June 12, 2000; 149(6): 1249 - 1262. [Abstract] [Full Text] [PDF]
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J. L. Alonso-Lebrero, J. M. Serrador, C. Dominguez-Jimenez, O. Barreiro, A. Luque, M. A. del Pozo, K. Snapp, G. Kansas, R. Schwartz-Albiez, H. Furthmayr, et al. Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells Blood, April 1, 2000; 95(7): 2413 - 2419. [Abstract] [Full Text] [PDF]
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M. Nieto, J. L. Rodriguez-Fernandez, F. Navarro, D. Sancho, J. M.R. Frade, M. Mellado, C. Martinez-A, C. Cabanas, and F. Sanchez-Madrid Signaling Through CD43 Induces Natural Killer Cell Activation, Chemokine Release, and PYK-2 Activation Blood, October 15, 1999; 94(8): 2767 - 2777. [Abstract] [Full Text] [PDF]
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M. Vicente-Manzanares, M. Rey, D. R. Jones, D. Sancho, M. Mellado, J. M. Rodriguez-Frade, M. A. del Pozo, M. Yanez-Mo, A. M. de Ana, C. Martinez-A., et al. Involvement of Phosphatidylinositol 3-Kinase in Stromal Cell-Derived Factor-1{alpha}-Induced Lymphocyte Polarization and Chemotaxis J. Immunol., October 1, 1999; 163(7): 4001 - 4012. [Abstract] [Full Text] [PDF]
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A. Al-Aoukaty, B. Rolstad, and A. A. Maghazachi Recruitment of Pleckstrin and Phosphoinositide 3-Kinase {gamma} into the Cell Membranes, and Their Association with G{beta}{gamma} After Activation of NK Cells with Chemokines J. Immunol., March 15, 1999; 162(6): 3249 - 3255. [Abstract] [Full Text] [PDF]
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