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,§
,§
Departments of
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Molecular Immunology,
Medicine, and
Flow Cytometry, Roswell Park Cancer Institute, Buffalo, NY 14263; and
§
Department of Hematology/Oncology, Arthur G. James Cancer Hospital and Research Institute, Ohio State University, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Moreover, CD56bright NK cells
bound with high efficiency to physiologic L-selectin ligands on
peripheral lymph node high endothelial venules (HEV). In sharp
contrast, CD56dim NK cells adhered poorly to HEV and were
predominantly L-selectin- or expressed L-selectin only at
low density. In CD56bright cells and a subpopulation of
CD56dim cells, L-selectin ligation by mAb cross-linking
activated lymphocyte function-associated Ag 1 (LFA-1), a second
adhesion molecule required for leukocyte extravasation. LFA-1 was
expressed on both NK subsets, although its density was constitutively
higher on CD56dim cells. Taken together, evidence of
differential expression of L-selectin and LFA-1 on
CD56bright and CD56dim NK subsets strongly
suggests unique migratory properties and functions of these cells
during the early immune response to foreign pathogens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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III). Approximately 10% of NK cells express CD56 at high
density (CD56bright CD16negative/dim),
while the more abundant NK cell subset expresses low levels of CD56
(CD56dim CD16bright) (3, 4, 5).
CD56bright NK cells are unique among unstimulated human
lymphocytes in their constitutive expression of the high affinity
heterotrimeric IL-2Rß (6, 7) and the c-kit tyrosine kinase
receptor (8). Thus, picomolar concentrations of IL-2 initiate a strong
proliferative response by CD56bright NK cells, which can be
potentiated by IL-15 or c-kit ligand (5, 6, 7, 8, 9, 10, 11). In contrast,
CD56dim NK cells do not express the c-kit receptor and
express only the intermediate affinity IL-2Rß (7, 8, 10).
Therefore, nanomolar concentrations of IL-2 are required to stimulate
CD56dim NK cell cytotoxicity, although these cells fail to
proliferate in response to IL-2 (5, 6, 7, 8, 9, 11). The CD56bright
NK subset also appears to produce significantly greater amounts of
IFN-, TNF-, and granulocyte/macrophage-CSF than
CD56dim NK cells following stimulation with IL-2 and IL-12,
even at nanomolar concentrations of IL-2 (12). Evidence that
CD56bright NK cells selectively respond to low
concentrations of lymphokines and monokines has led to the hypothesis
that this NK cell subset has a distinct role in the early stages of the
immune response when these cytokines are present in limiting
concentrations (3, 13).
NK cells are localized in high numbers in the spleen, where they have
ready access to foreign pathogens. In addition, there is increasing
evidence that NK cells can be actively recruited to peripheral tissues
at sites of infection, injury, or tumor growth (14, 15, 16, 17, 18, 19). Leukocyte
emigration from the blood into tissues is dependent on a multistep
adhesion cascade (20, 21). The L-selectin adhesion molecule (CD62L) on
the surface of B and T lymphocytes, neutrophils, and monocytes mediates
the initial attachment and slow rolling of these cells along the
luminal surface of specialized high endothelial venules
(HEV),3 a crucial first step
in the extravasation of immune effector cells at sites of inflammation
or injury as well as in lymph nodes and Peyer’s patches. Firm adhesion
and transendothelial migration of lymphocytes are dependent on the
interaction of the leukocyte integrin LFA-1 with ICAM-1 and ICAM-2 on
endothelial cells. Recent studies have shown that L-selectin engagement
by mAb or a physiologic ligand, GlyCAM-1 (glycosylation-dependent cell
adhesion molecule-1) up-regulates LFA-1 affinity in neutrophils and T
lymphocytes (22, 23, 24), suggesting that the L-selectin pathway functions
as an intravascular trigger to facilitate leukocyte extravasation. The
4ß1 and 4ß7
integrins have also been implicated in recruitment of lymphocytes to
tissues through binding to endothelial counterreceptors, VCAM-1 and
MAdCAM-1 (mucosal vascular addressin-1) (20, 21).
NK cells adhere to and transmigrate across endothelial cells in vitro
at a faster rate than T cells (25, 26, 27), supporting the purported role
of these immune effector cells during the early stages of an immune
response before mobilization of Ag-restricted T lymphocytes. The
contribution of L-selectin to NK cell homing has not been fully
resolved, in part, because of the wide discrepancy among reports with
respect to the constitutive level of L-selectin expression on
circulating NK cells. In this regard, the reported frequency of
peripheral blood NK cells expressing L-selectin has ranged from 10 to
60% (5, 28, 29, 30, 31, 32). Possible explanations for these conflicting results
include 1) heterogeneous expression of L-selectin by NK cell subsets,
as suggested previously (5, 29), 2) variability in L-selectin
expression among different donor NK cell populations, and 3) modulation
of L-selectin levels during lymphocyte isolation from peripheral blood.
The relationship between L-selectin expression and function on
CD56bright and CD56dim NK cell subsets has not
been previously addressed. Moreover, although LFA-1 and
4ß1/7 integrins have been strongly
implicated in NK cell recruitment to tissues and NK cytolytic function
(25, 27, 33, 34), the relative level of expression of these molecules
by NK cell subsets has not been investigated.
In this report, we have examined the expression and function of leukocyte homing receptors on NK cell subsets. These studies demonstrate for the first time differential utilization of the L-selectin adhesion pathway by CD56bright and CD56dim NK cells. L-selectin was found to be expressed at the highest density on CD56bright NK cells relative to other peripheral blood leukocyte subsets, including CD56dim NK cells, T cells, B cells, neutrophils, and monocytes. Consistent with these observations, CD56bright NK cells exhibit highly efficient L-selectin-dependent adhesion to HEV in lymph node tissue sections when compared with the CD56dim NK subset. Moreover, mAb-induced L-selectin ligation was shown to increase LFA-1 function in the CD56bright NK cell subset as well as in a subset of CD56dim NK cells. Taken together, these results predict that the CD56bright NK cell subset has a selective advantage, compared with CD56dim NK cells, in extravasating across HEV via the L-selectin pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated from normal donor buffy coat leukocyte concentrates (American Red Cross, Buffalo, NY) by Ficoll/Hypaque centrifugation as described (8, 35, 36). Following removal of adherent cells, the PBL population was enriched for NK cells by depleting T cells, B cells, and the remaining monocytes using goat anti-mouse Ig-conjugated immunomagnetic beads (Per Septive Biosystems, Framinghom, MA), and a combination of murine mAb reactive against human CD3, CD4, and HLA-DR (8). NK-enriched populations typically contained 80 to 90% CD56+ cells. PBL and NK-enriched populations were cultured at a final concentration of 4 x 106 cells/ml in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% FCS (Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin, and 50 µg/ml streptomycin.
Murine 3T3 cells stably transfected with human ICAM-1 and 3T3-Neo controls have been described previously (36, 37) and were generously provided by Dr. W. Muller (Rockefeller University, New York, NY). These cells were maintained in culture in DMEM (Life Technologies) with 10% FCS, 1 mg/ml G418 (Life Technologies), and 0.4 mg/ml hygromycin (Calbiochem, La Jolla, CA). The ICAM-1 transfectant population was >95% positive for ICAM-1. 3T3-Neo controls were >99% ICAM-1-negative.
Cytokines, mAb, and reagents
The following recombinant human cytokines were used: IL-2
(Hoffmann-La Roche, Nutley, NJ; sp. act., 1.53 x
107 U/mg); IL-10 (Schering-Plough, Kenilworth, NJ; sp.
act., 11.0 x 106 IRU/mg); IL-12 (Genetics Institute,
Andover, MA; sp. act., 4.5 x 106 U/mg); IL-15
(Immunex Corp., Seattle, WA); TNF- (Ashahi Chemical, Tokyo, Japan;
sp. act., 2 x 103 U/mg); IFN- (Shering-Plough; sp.
act., 2.2 x 108 IU/mg); and TGF-ß2 (R&D Systems,
Minneapolis, MN). The L-selectin-specific mAb DREG-56 (38) was a
generous gift from Dr. E. Butcher (Stanford University, Stanford, CA).
Anti-ß1-integrin mAb was kindly provided by Dr. Richard
Bankert (Roswell Park Cancer Institute, Buffalo, NY). The
anti-HLA-DR mAb has been described previously (8). The following
mAb were obtained commercially: L-selectin-specific mAb Leu-8-FITC,
anti-CD3 (Leu-4)-PE, anti-CD16-PE and FITC- or phycoerythrin
(PE)-conjugated isotype-matched control mAb (Becton Dickinson
Immunocytometry, San Jose, CA); anti-CD56-PE and
L-selectin-specific TQ1 mAb (Coulter Immunology, Hialeah, FL);
FITC-labeled anti-human LFA-1 (CD11a)-FITC (Endogen, Boston,
MA); unconjugated anti-LFA-1 (TS1/22), anti-CD3 (OKT3) and
anti-CD4 (OKT4) (American Type Culture Collection, Manassas, VA);
anti-CD49d-FITC (4-integrin) (Immunotech, Westbrook,
ME); and anti-CD64-TriColor and anti-CD3-TriColor (Caltag,
Burlingame, CA). Goat-anti-mouse IgG-RITC (rhodamine
isothiocyanate) was obtained from Southern Biotechnology (Birmingham,
AL) and sheep anti-mouse IgG was from Organon Teknika (West
Chester, PA). PMA and fibronectin were purchased from Sigma Chemical
(St. Louis, MO).
Flow cytometric analysis
Enriched PBL or NK cell populations were analyzed for
simultaneous two-color or three-color immunofluorescence expression of
adhesion molecules (L-selectin, LFA-1, and 4- or
ß1-integrins) and either CD56 (NK cells) or CD3 (T cells)
as described (8, 35). For analysis of leukocyte-enriched whole blood,
samples were washed twice with PBS containing 10 U/ml heparin,
incubated with goat serum for 10 min at 4°C to block Fc receptor
sites, and then incubated for 15 min simultaneously with FITC-labeled
mAb specific for adhesion molecules and PE-labeled lymphocyte-specific
mAb. RBC were lysed (150 mM ammonium chloride, 10 mM potassium
bicarbonate, 97 µM EDTA) before fixation in 1% formaldehyde/PBS.
Background fluorescence was determined on cells stained with
fluorochrome-labeled isotype-matched nonreactive mAb. A total of 5000
events were collected for each leukocyte population on a FACScan
(Becton Dickinson) in the Roswell Park Flow Cytometry facility, and
analysis was performed on WinList 1.0 (Verity Software House, Topsham,
ME). T cells or NK cell subsets were analyzed using a lymphocyte gate;
the FL-2 amplifier gain was adjusted to trigger selectively on either
CD3+, CD56dim, or CD56bright cells.
Monocytes and neutrophils were discriminated by forward and side
scatter parameters and on the basis of CD16 (neutrophils) or CD64
(monocytes) fluorescence.
Phosphomonoester core polysaccharide (PPME) binding assay
Analysis of L-selectin-dependent binding of lymphocytes to PPME, the phosphomonoester core from Hansenula hostii phosphomannan, was performed as described (35, 39, 40). Enriched NK cell populations were initially stained with CD56-PE, washed once in RPMI 1640 medium, resuspended at 5 x 106 cells/ml, and then incubated for 15 min at 4°C either in medium alone or with the L-selectin-specific TQ1 blocking mAb (10 µg/ml). Without washing, cells were then incubated with a 1:200 dilution of fluorescein-conjugated PPME (generous gift of Dr. L. Stoolman, University of Michigan, Ann Arbor, MI) for 30 min at 4°C and analyzed immediately by flow cytometry.
Quantitative lymphocyte adhesion assay
Lymphocyte binding to HEV was assessed essentially as described with the following modifications (35, 36, 41). Enriched NK cell populations were initially stained at 4°C with anti-CD56-PE primary mAb and goat anti-mouse IgG-RITC secondary Ab to permit analysis of NK cell subset adhesion to HEV. In pilot experiments, it was determined that identical numbers of cells were bound to HEV regardless of whether CD56 was fluorescently labeled on NK cells. NK-enriched populations or PBL were then washed and resuspended at 5 x 107 cells/ml in RPMI 1640 medium containing 10% FCS and incubated for 30 min at room temperature with or without saturating amounts of L-selectin-specific blocking mAb (DREG-56). Lymphocytes (5 x 106 cells in 100 µl) were overlaid onto 12-µm-thick cryosections of BALB/c lymph nodes mounted on glass slides. Previous studies have established that L-selectin binding specificity is maintained during assay of human lymphocyte adhesion to mouse lymph node HEV (38). Slides were rotated at 112 rpm (Labline Instrument, Melrose Park, IL) at 4°C for 30 min, and nonadherent cells were removed by gentle washing in cold PBS. Slides were fixed vertically in 3% formaldehyde (Ernest F. Fullam, Catham, NY)/PBS for 1 h, then either rinsed in PBS (in the case of NK-enriched samples) or permeabilized in 70% ethanol and stained with 0.5% toluidine/absolute ethanol (PBL samples). Adhesion of CD56bright and CD56dim NK cells was quantified under double blind conditions using an Olympus BH2-RFL fluorescence microscope (Olympus Optical, Tokyo, Japan). PBL adhesion to HEV was evaluated by light microscopy. A total of 300 to 500 HEV were examined, and data are expressed as the mean number of lymphocytes bound per HEV ± SD; each sample was quantified in triplicate.
LFA-1-dependent cell adhesion assay
LFA-1-dependent adhesion of PBL to ICAM-1-transfected 3T3-fibroblasts was evaluated essentially as described (36, 37), with the following modifications to allow the discrimination of adherent NK cell subsets. Immediately before initiation of the assay, 2 x 106 enriched-NK cells were stained with anti-CD56 mAb and goat anti-mouse IgG-RITC secondary Abs. L-selectin cross-linking was performed by incubating cells with 5 µg/ml of DREG-56 mAb (30 min, room temperature), followed by 10 µg/ml sheep anti-mouse IgG (2 h at 37°C). In parallel samples, cells were incubated either in RPMI 1640 medium/10% FCS alone or in the presence of 100 ng/ml PMA to activate LFA-1 function. Cells were then washed and incubated in the absence or presence of saturating concentrations of anti-LFA-1 blocking mAb (TS1/22) for 30 min at 37°C. A total of 106 cells (1 ml) were added to confluent monolayers of ICAM-1-transfected 3T3 cells or Neo transfectants grown on fibronectin-coated tissue culture chamber slides (Miles Laboratories, Naperville, IL). Following incubation for 30 min at 37°C, nonadherent cells were removed by gentle washing, and adherent cells were fixed in 3% formaldehyde. The number of adherent CD56+ NK cells was quantified in five low power fields (20x) in replicate by fluorescence microscopy. Since the CD56 Ag became capped on the cell surface under these conditions, it was not possible to visually distinguish CD56bright from CD56dim cells by conventional fluorescence microscopy. Therefore, the proportion of CD56bright:CD56dim NK cells was determined by quantitating the fluorescence intensity of single cells by confocal fluorescence microscopy (MRC 600 CSLM; Bio-Rad, Palo Alto, CA). A total of 100 cells was examined at random from each condition, and total fluorescence was determined by electronic integration of capped regions of fluorescence using COMOS 6.03 (Bio-Rad).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A comparative analysis of adhesion molecule expression on NK cell subsets was performed by multiparameter flow cytometry using unseparated whole blood to avoid modulation of the cell surface levels of these molecules during the isolation of leukocyte subpopulations. Several striking differences were detected in L-selectin and LFA-1 expression by CD56bright and CD56dim peripheral blood NK cells that are suggestive of a differential ability of these cells to extravasate across HEV in lymphoid tissues and inflamed tissues.
In 20 donors examined, L-selectin was universally found to be highly
expressed on the CD56bright NK cell subset (Figs. 1
and 2). Notably, the
density of L-selectin on this NK cell subset was markedly higher than
on CD56dim NK cells, T cells, B cells (not shown),
neutrophils, or monocytes. In sharp contrast, in the majority of donors
examined, L-selectin was expressed only on a minor proportion of
CD56dim NK cells (<25%) at a density considerably lower
than on CD56bright NK cells (Fig. 1 and 2A). However, in
30% of donors, a different pattern of L-selectin expression was
observed on CD56dim NK cells, indicating that considerable
variability exists among donors with regard to the constitutive levels
of L-selectin on this NK cell subset. In these individuals, L-selectin
was detected on up to 50% of CD56dim NK cells, albeit at a
low density compared with CD56 bright NK cells or T cells
(Fig. 2B). Discrimination of CD56+ NK
cells from the CD56+CD3+ T cell subset that is
present in peripheral blood (42) by three-color flow cytometric
analysis further revealed that both L-selectin and CD56 were
consistently expressed at a lower density on this T cell subpopulation
than on CD56dim NK cells (n = 15
donors, data not shown). L-selectin levels on either NK subset remained
unchanged during their isolation from whole blood or after culture
for 1 day (not shown).
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). These results are consistent with the high
expression of LFA-1 previously described on total NK cell populations
(34). The density of LFA-1 detected on CD56bright NK cells
was comparable with T lymphocytes and neutrophils. No difference was
observed in the expression of the 4-integrin on NK cell
subsets. ß1-integrins were consistently found to be
expressed at a moderately higher density on the CD56bright
NK subset when compared with CD56dim cells. PMA induces L-selectin down-regulation in NK cell subsets
L-selectin is rapidly shed from the plasma membrane of T and B
lymphocytes, neutrophils, and monocytes in direct response to
stimulation of protein kinase C by phorbol esters (i.e., PMA), although
the kinetics vary in different leukocyte populations (20, 35, 38).
Therefore, it was of interest to examine the effects of PMA on the
kinetics of L-selectin down-regulation in CD56bright
and CD56dim NK cells. Following incubation of
leukocyte-enriched whole blood with PMA, a gradual decrease in
L-selectin surface levels occurred over the course of 1 h in the
CD56bright and CD56dim NK cell subsets (Fig. 3). The kinetics of L-selectin
down-regulation in NK cells closely paralleled the PMA response of T
lymphocytes (Fig. 3) and B lymphocytes (data not shown). These
observations contrast sharply with the rapid loss of L-selectin
detected within 1 min following PMA activation of neutrophils and
monocytes.
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NK cell expansion and function is regulated by numerous
immunomodulatory cytokines including IL-2, IL-10, IL-12, IL-15, and
TGF-ß (1, 2, 6, 7, 8, 9, 11, 43, 44). Lymphocyte activation in response to
IL-2, mitogens, or CD3 cross-linking has been shown to down-regulate
L-selectin cell surface levels on T cells and total NK cell populations
(28, 30, 32, 35, 45), although the effects of IL-2 or other cytokines
on L-selectin expression by CD56bright and
CD56dim NK cell subsets has not been previously examined.
Moreover, the effect of proinflammatory cytokines that are present in
tissues during acute inflammation or injury (e.g., TNF-, IFN-) on
L-selectin expression by NK cell subsets is not known. To address these
issues, enriched populations of NK cells were cultured for 24 h in
the presence of recombinant cytokines, and the expression of L-selectin
on NK cell subsets was determined by two-color flow cytometric analysis
(Fig. 4). IL-12 caused a marked increase
in L-selectin expression on both CD56bright and CD56
dim NK cell subsets, as indicated by an increase in both
L-selectin density (i.e., indicated by the mean channel fluorescence)
and the frequency of L-selectin-positive cells. A moderate increase in
L-selectin density was also observed in response to IL-10 (Fig. 4) and
IFN- in both NK cell subsets (data not shown). In contrast,
L-selectin levels were markedly down-regulated in the
CD56bright NK cell subset following culture with IL-2,
IL-15, or TGF-ß. Although IL-2, IL-15, or TGF-ß did not cause a
major change in the CD56dim subset in terms of the
frequency of L-selectin-positive cells or L-selectin density, (as would
be expected since these values are heavily weighted by the large
L-selectin-negative population), a moderate decrease in fluorescence
intensity was detected in response to these cytokines (note the
downward shift on the log scale). TNF- had essentially no effect on
L-selectin expression by NK cell subsets.
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Studies were performed to determine whether the high density of
L-selectin detected on CD56bright NK cells
corresponded with efficient binding of this NK cell subset to soluble
L-selectin carbohydrate ligands, i.e, PPME, and to physiologic ligands
expressed on peripheral lymph node HEV. These functional assays are
highly predictive of the L-selectin-dependent homing potential of
leukocytes (20, 21). For these studies, an enriched population of NK
cells (>90% CD56+CD3-) was used in which
91% of CD56bright NK cells expressed high levels of
L-selectin and 20% of CD56dim cells expressed this
adhesion molecule at low density (i.e., the NK cells from this donor
were phenotypically similar to the NK cell subsets shown in Fig. 1).
The data shown in Figure 5 indicate that
CD56bright NK cells bound high levels of PPME in an
L-selectin-dependent manner that was strongly inhibited by the
L-selectin-specific TQ1 mAb, whereas the majority of the
CD56dim NK population bound low to non-detectable amounts
of PPME.
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, an enriched population of NK cells
comprised of 75% CD56dim CD3negative cells and
10% CD56bright cells was initially labeled at 4°C with
anti-CD56 primary mAb and goat-anti-mouse IgG-RITC to allow the
discrimination of these NK cell subsets in the adhesion assay.
L-selectin was expressed on >98% of the CD56bright cells
and on <25% of the CD56 dim NK cells of this particular
donor. Cells were then overlaid onto frozen sections of murine lymph
nodes, and adherence of CD56bright and CD56dim
NK cells to specialized HEV within tissues was quantified by
fluorescence microscopy. Despite the fact that CD56bright
NK cells were present in low numbers in the initial NK-enriched
population, this NK cell subset represented a high proportion (>90%)
of NK cells bound to HEV in a manner that could be inhibited
specifically by the L-selectin-blocking mAb, DREG-56. In contrast, only
low levels of L-selectin-dependent adhesion of CD56dim NK
cells to lymph node HEV were detected. Parallel analysis of PBL
adhesion to HEV indicated that comparable numbers of PBL and
CD56bright NK cells bound to HEV (Fig. 6), further
indicating that this numerically limited NK cell subset is capable of
highly efficient adhesion to HEV under shear conditions.
Immunofluorescence microscopy revealed that >70% of bound PBL were
CD3+ T cells (data not shown).
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Previous studies have demonstrated that Ab-mediated cross-linking
of L-selectin on neutrophils and T lymphocytes up-regulates the
function of ß2-integrins (22, 23, 24, 46), suggesting
that cooperation between distinct leukocyte-endothelial cell adhesion
events facilitate leukocyte extravasation. To address whether
L-selectin also stimulates NK cell adhesion through a
ß2-integrin pathway, an enriched population of NK cells
(>90% CD56+CD3-) was initially labeled with
anti-CD56 mAb and goat-anti-mouse IgG-RITC secondary Ab to allow
identification of NK cell subsets. The frequency of
CD56bright and CD56dim cells expressing
L-selectin in the representative experiment shown in Figure 7 and Table I was 94% and 18%, respectively, while
the ratio of CD56bright to CD56dim cells that
expressed L-selectin was 1:2.4. Following stimulation of cells under
various conditions, LFA-1-dependent adhesion of
fluorochrome-labeled CD56+. NK cells to monolayers of
ICAM-1-transfected 3T3 cells or Neo-transfectant control cells was
quantified by fluorescence microscopy. The CD56bright and
CD56dim NK cell subsets could not be distinguished visually
by conventional fluorescence microscopy, since a majority of the
CD56 Ag became capped during the adhesion assay. Therefore, the
fluorescence intensity of individual CD56-positive cells was quantified
by confocal fluorescence microscopy and computer assisted digital
analysis (Table I).
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), consistent with the reported constitutive low
affinity of LFA-1 on circulating leukocytes (20, 21). Thus, initial
CD56 labeling did not directly activate LFA-1 function. Following
stimulation with PMA, a known activator of LFA-1 affinity (20, 21), a
threefold increase in LFA-1-dependent NK cell adhesion to ICAM-1
transfectants was detected. PMA-stimulated NK cell adhesion to ICAM-1
transfectants was strongly inhibited by an LFA-1 function-inhibiting
mAb. Notably, despite higher expression of LFA-1 by CD56dim
cells (Fig. 1), the proportions of CD56bright and
CD56dim cells present in the initial NK-enriched population
and in PMA-activated adherent NK cells were similar (Table I),
indicating that preferential activation of LFA-1 activity does not
occur in either NK cell subset in response to phorbol ester
stimulation. L-selectin cross-linking via DREG-56 mAb stimulated a
threefold increase in LFA-1-dependent adhesion of NK cells to ICAM-1
transfectants (Fig. 7). While L-selectin ligation increased
LFA-1-dependent adhesion of both CD56bright and
CD56dim NK cells (combined data in Fig. 7 and Table I),
considerable enrichment of CD56bright NK cells in the
adherent population was observed (i.e., CD56bright cells
comprised 37% of adherent NK cells in DREG-56-stimulated cultures
compared with 12% of the initial NK-enriched population), signifying
highly efficient L-selectin-mediated activation of LFA-1 function in
this numerically rare NK cell subset. Notably, the proportion of
CD56bright cells to CD56dim cells in the
adherent populations (i.e., 1:2.7, Table I) closely paralleled the
ratio of these subsets that expressed L-selectin in the initial
populations (i.e., 1:2.4), suggesting that LFA-1 activation by DREG-56
mAb occurred selectively in L-selectin+ NK cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Data presented in this report demonstrate for the first time a direct correlation between L-selectin expression and function in CD56bright and CD56dim NK subsets, suggesting distinct migratory pathways of these leukocytes. L-selectin was shown to be highly expressed on resting CD56bright NK cells, at a greater density than on all other peripheral blood leukocytes. These data are consistent with the high frequency of L-selectin expression noted on CD56bright (CD16negative or CD16dim NK) cells in two previous reports (5, 29). In the current study, the CD56bright subset was further shown to bind with high efficiency to physiologic L-selectin ligands on specialized HEV. In sharp contrast, CD56dim NK cells bound poorly to HEV and were predominantly L-selectin-negative or expressed L-selectin only at low density. Based on the significant enrichment of CD56bright cells in the HEV-adherent NK fraction, of which >90% represented the CD56bright subset, it appears that the low density of L-selectin on CD56dim cells is not sufficient to optimally mediate adhesion to HEV under physiologic shear conditions. Taken together, these results suggest a model whereby L-selectin preferentially facilitates trafficking of the CD56bright NK cell subset from the blood into secondary sites during the early phase of infection or injury.
While L-selectin was found to be consistently expressed at high levels on the CD56bright cells, considerable heterogeneity was observed among donors in L-selectin expression on the numerically major CD56dim NK subset. These results may explain the variable findings of several studies regarding the constitutive level of L-selectin expression on NK cells in which the contributions of CD56bright and CD56 dim cells were not determined (28, 30, 31, 32). The reason for the variability in L-selectin expression on CD56dim cells is not known but may reflect differences among donors in the activation status of circulating NK cells. While healthy donors were used in these studies, subclinical or trivial infections could have lead to variations in L-selectin expression. As indicated in the present study, lymphocyte activation (i.e., in response to PMA or stimulatory cytokines such as IL-2) leads to rapid loss of L-selectin surface expression (20, 35, 38). In addition, heterogeneity in L-selectin expression may reflect the differentiation stage of circulating NK cells such that in some individuals, a higher percentage of the mature CD56dim population (5, 7, 13) transiently maintains some of the phenotypic characteristics (i.e., L-selectin levels) of the less mature CD56bright subset.
Emerging evidence that the CD56bright NK cell subset is unique in its high constitutive expression of multiple adhesion molecules that mediate cell-cell and cell-matrix interactions, such as L-selectin (this report and Refs. 5 and 29), CD2, CD44, CD11c, and ICAM-1 (5, 29, 34), suggests that this NK subset circulates in a heightened state of functional readiness. L-selectin potentially functions at several discrete levels to recruit CD56bright NK cells to secondary sites. In addition to mediating leukocyte-endothelium binding, L-selectin has recently been shown to support leukocyte-leukocyte interactions involving neutrophils (47, 48), suggesting similar functions for L-selectin in lymphocyte population such as CD56bright NK cells. The ability of L-selectin to mediate leukocyte rolling on adherent neutrophil monolayers has been proposed as a mechanism to provide access of circulating immune effector cells to tissues in the early stages of an immune response when newly recruited neutrophils effectively form a barrier to underlying endothelium (47, 48).
L-selectin was also shown in the present study to regulate LFA-1 function in NK cells, similar to its reported function in neutrophils and T lymphocytes (22, 23, 24, 46, 49). LFA-1 plays a crucial role in strengthening leukocyte-endothelial adhesion and promotes transendothelial migration (20, 21). Notably, recent studies by Berman et al. have demonstrated that CD56bright NK cells migrate at a significantly faster rate across cytokine-activated endothelium than CD56dim NK cells (37), although the adhesion pathway was not identified in this prior study. Our data demonstrate that the DREG-56 mAb, used in place of L-selectin ligand, strongly stimulates LFA-1 activation in CD56bright cells as well as a subset of CD56dim cells, presumably the CD56dim/L-selectindim population. However, in a physiologic setting in which L-selectin adhesion to HEV ligands is expected to occur preferentially in CD56bright cells, L-selectin-mediated activation of LFA-1 may have only a limited role in the emigration of the CD56dim subset. CD44 and CD11c are also present at a higher surface density on CD56bright NK cells than on the CD56dim subset (5, 29). These adhesion molecules mediate leukocyte adhesion to vascular endothelium and to extracellular matrix proteins, i.e., hyaluronate and fibrinogen, and thus, potentially function to facilitate NK cell extravasation and migration through the interstitial matrix in tissues (21). Within tissues, high constitutive levels of ICAM-1 on the CD56bright NK subset (5, 29) could mediate interactions with accessory cells such as monocytes via binding to its ligands LFA-1 and Mac-1.
The selective migration of the CD56bright NK subset via
L-selectin into inflammatory sites may be advantageous at the
initiation of infection, when cytokine levels are low but sufficient to
cause proliferation of this subset. CD56bright NK cells are
unique among circulating lymphocytes because of their constitutive
expression of the high affinity IL-2R (6, 7). Thus, limited
concentrations of T cell-derived IL-2 in tissues before extensive T
cell infiltration would theoretically favor proliferation of
CD56bright NK cells over other unstimulated lymphocyte
populations. Recent evidence (9) that the monocyte-derived cytokine
IL-15, which also binds to the IL-2ßR, stimulates
CD56bright NK cell proliferation is particularly
intriguing, since monocytes are known to function in the earliest phase
of the immune response to bacterial or viral pathogens. Two
monocyte-produced cytokines, IL-12 and IL-15, have been shown to act
synergistically to stimulate NK cell production of proinflammatory
cytokines including IFN-, TNF-, and granulocyte/macrophage-CSF
(1, 2, 9, 11, 13, 50, 51), suggesting a paracrine loop in which NK
cell-produced cytokines regulate monocyte differentiation and effector
function. Significantly, the CD56bright NK subset produces
markedly higher levels of IFN- than CD56dim cells
following stimulation by IL-12, in combination with either IL-15,
IL-1ß, or TNF- (12).
NK-derived cytokines may also recruit other leukocyte types to the
site. The role of IFN- and TNF- in stimulating endothelial cell
expression of ICAM-1, VCAM-1, and E-selectin is well established (20, 21). Thus, high level production of these cytokines by
CD56bright NK cells early in the immune response feasibly
potentiates the recruitment of T lymphocytes, CD56dim NK
cells, and monocytes to tissues. The high density of LFA-1 on
CD56dim NK cells demonstrated in this report may facilitate
extravasation of this immune effector subset through interactions with
ICAM-1 on cytokine-activated endothelium. Alternatively, high density
LFA-1 may potentiate the cytolytic effector function of NK cells, as
suggested previously (34). In addition, production by
CD56bright NK cells of the C-C chemokine MIP-1
(macrophage inflammatory protein-1) in response to IL-12 in
combination with IL-15, as described recently (52), could enhance
immune effector cell migration within inflammatory sites as well as
their emigration from the blood.
In this report, L-selectin was shown to be down-regulated
following stimulation of CD56bright and CD56dim
NK cells by IL-2, IL-15, TGF-ß, or PMA in vitro. Similar changes in
L-selectin expression in response to IL-2 or PMA have been described
for total NK cell populations (28, 30, 32). These data suggest that
L-selectin would be rapidly lost on activated CD56bright
cells shortly after their infiltration into tissues and activation in
situ, although it is difficult to predict the outcome in tissues
containing multiple cytokines that exert opposing effects on L-selectin
expression (e.g., IL-12, IL-10, IFN- vs IL-2, IL-15, and TGF-ß).
In this regard, we have found in preliminary studies that inclusion of
IL-12 in IL-15-stimulated cultures partially protects L-selectin
expression on the CD56bright NK cell population
(n = 2). The observed effect of cytokines on
L-selectin expression by NK cells may have important implications for
clinical studies in which cytolytic NK cells are expanded in vitro by
IL-2 before use in adoptive immunotherapy in cancer patients. Under
these conditions, cytokine-activated NK cells would likely be excluded
from tissues requiring L-selectin in the extravasation process. Further
studies are required to determine the value of using cytokines such as
IL-12, IL-10, or IFNs, as suggested here and in previous studies (40, 53), to restore L-selectin levels on expanded populations of immune
effector cells immediately before their transfer to patients.
In conclusion, data presented in this report strongly implicate L-selectin in the dissemination of the CD56bright NK cell subset to tissues in the early phase of an immune response to foreign pathogens. Based on these results, we speculate that the L-selectin adhesion pathway is involved in the rapid extralymphoid accumulation of NK cells as described previously at sites of viral infection (1, 2, 15, 16, 17), inoculation with bacterial agents (14), allograft rejection (18), or tumor growth (19). The finding that CD56bright NK cells bind efficiently to well-established sites of L-selectin ligand on HEV within lymph node tissues further raises the possibility that this NK cell subset, like naive T lymphocytes (20, 21), recirculates through peripheral lymphoid tissues as part of physiologic immune surveillance mechanisms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sharon S. Evans, Roswell Park Cancer Institute, Buffalo, NY 14263. E-mail address:
3 Abbreviations used in this paper: HEV, high endothelial venule; PPME, phosphomonoester core polysaccharide; RITC, rhodamine isothiocyanate; PE, phycoerythrin; LFA-1, lymphocyte function-associated Ag 1.
Received for publication November 12, 1997. Accepted for publication February 24, 1998.
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