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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2976-2983
Molecular Mechanisms of Zinc-Dependent Leukocyte Adhesion Involving
the Urokinase Receptor and  2-Integrins
By
Triantafyllos Chavakis,
Andreas E. May,
Klaus T. Preissner, and
Sandip M. Kanse
From the Haemostasis Research Unit, Kerckhoff-Klinik, MPI, Bad
Nauheim; and the Medizinische Klinik und Deutsches Herzzentrum,
Technische Universität München, Munich, Germany.
 |
ABSTRACT |
The trace element Zinc (Zn2+) has been implicated as a
mediator in host defense, yet the molecular basis for its extracellular functions remains obscure. Here, we demonstrate that Zn2+
can induce the adhesion of myelomonocytic cells to the endothelium, as
well as to the provisional matrix proteins vitronectin (VN) and
fibrinogen (FBG), which are pivotal steps for the recruitment of
leukocytes into inflamed/injured tissue. Physiologic concentrations of
Zn2+ increased the urokinase receptor (uPAR)-mediated
adhesion of myelomonocytic cells to VN, whereas other divalent cations
had smaller effects. Zn2+-induced cell adhesion to VN was
abolished by cation chelators such as 1-10-phenanthroline, as well as
by plasminogen activator inhibitor-1 (PAI-1) and a monoclonal antibody
(MoAb) against uPAR. These characteristics could be recapitulated with
a uPAR-transfected cell line emphasizing the specificity of this
receptor system for Zn2+-dependent cell adhesion. Like
urokinase (uPA), Zn2+ increased the binding of
radiolabeled VN to uPAR-expressing cells, as well as the interaction of
VN with immobilized uPAR in an isolated system. Moreover,
Zn2+ enhanced leukocytic cell adhesion to FBG and
endothelial cell monolayers by activating  2-integrins.
Instead of the direct 2-integrin activation through the
divalent cation binding site, Zn2+-induced integrin
activation was mediated via uPAR, a crucial regulator of this system.
The present study uncovers for the first time
Zn2+-mediated cell adhesion mechanisms that may play a
crucial role in modulating leukocyte adhesion to vessel wall components.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
WHEN LEUKOCYTES EMIGRATE from the
bloodstream into sites of inflammation or injury, they undergo a
complex sequence of adhesion and locomotion steps. These highly
coordinated processes require the expression and upregulation of
various adhesion receptors on the surface of leukocytes and vascular
cells.1,2 During their transmigrational phase, leukocytes
adhere to provisional matrix substrates such as fibrinogen (FBG) and
vitronectin (VN), which become deposited at these sites upon increases
in vascular permeability or damage.3 Both adhesion proteins
are recognized by different, temporally activated receptors:
 M2-integrin (Mac-1, CD11b/CD18) mediates interaction
with FBG, which is profoundly affected by extracellular divalent
cations,4,5 whereas VN serves as a ligand for the urokinase
receptor (uPAR) in a divalent cation-independent manner.6,7
The VN-uPAR interaction mediates cell adhesion and is augmented by uPA,
but blocked by plasminogen activator inhibitor-1 (PAI-1).6
uPAR thereby serves a dual role in cellular interactions, (1) being
critical for pericellular proteolysis and (2) contributing to cell
adhesion in a proteolysis-independent fashion.8,9
Consequently, the expression level of uPAR on various cell types
correlates with their migratory and invasive potential.10
Moreover, independently of its enzymatic activity, uPA stimulates
monocytic cell chemotaxis11,12 and regulates adhesion of
neutrophils and monocytes.13,14 For some of these functions, uPAR is suggested to use integrins as adapters, since it is
able to form complexes with 2-integrins15-17
or interferes with 1-integrin ligation.18
During our investigations of monocyte cell-matrix interactions, we
noted a profound effect of zinc (Zn2+) on cell adhesion.
The trace element Zn2+ is an important cofactor of several
proteins and enzymes, such as transcription factors,19,20
focal adhesion molecules,21 or matrix
metalloproteinases.22 Recently, Zn2+ has been
shown to promote the interaction of uPAR and kininogen and this is
inhibited by VN, which suggests a role for Zn2+ in
regulating cell adhesion.23 Zn2+ has also been
reported to enhance interleukin-1 and tumor necrosis factor-
levels in peripheral blood mononuclear cells,24,25 to
influence ICAM-1 expression,26 or to increase neutrophil adhesion.27
The observations that Zn2+ deficiency is associated with
skin lesions, impaired wound healing, and cell-mediated immune
disorders28-30 prompted us to investigate the effect of
Zn2+ on adhesion of leukocytic cells in more detail. The
results indicate that the adhesion of these cells to provisional matrix
proteins VN and FBG and to the endothelium is stimulated by
Zn2+ in a uPAR-dependent manner.
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MATERIALS AND METHODS |
Reagents.
Recombinant Gly158scuPA (noncleavable mutant of high molecular weight
uPA)31 was kindly obtained from Dr H.R. Lijnen (University of Leuven, Belgium) and uPA was from Medac (Hamburg, Germany). VN was
purified from human plasma and converted to the multimeric form as
previously described.32,33 FBG was purchased from
Kabivitrum (Munich, Germany), and recombinant uPAR was kindly provided
by Dr N. Behrendt (Finsen Laboratory, Copenhagen, Denmark). MoAb 13H1
against human VN33 was provided by Dr P. Declerck
(University of Leuven, Belgium), and anti-uPAR MoAb R3 was given by Dr
G. Hoyer-Hansen (Finsen Laboratory, Copenhagen, Denmark). The blocking anti-2-integrin MoAb 60.3 was obtained from Dr J. Harlan
(University of Washington, Seattle, WA); MoAb 24, which recognizes an
activation-dependent epitope of 2-integrin subunit, was
kindly provided by Dr N. Hogg (University of London, UK), MoAb 2LPM19c
was from Dako (Hamburg, Germany), and the blocking MoAb, Game 46, against mouse 2-integrins was from Pharmingen (Hamburg,
Germany). Bovine serum albumin (BSA), ZnCl2,
MnCl2, FeCl2, NiCl2,
CuCl2, and CoCl2 were from Sigma (Munich, Germany), 1-10-phenanthroline was from Fluka (Neu-Ulm, Germany), vitamin D3 was from Biomol (Hamburg, Germany), transforming
growth factor- was from R&D Systems (Boston, MA), and interleukin-3 was from PBH (Hannover, Germany).
Cell culture.
Human myelomonocytic cells U937 and HL60 were from American Type
Culture Collection (ATCC; Rockville, MD) and cultured as described by
the supplier in RPMI-1640 medium containing 10% (vol/vol) fetal calf
serum. BAF-3 cells were from ATCC and cultured in RPMI-1640 medium
containing 10% (vol/vol) fetal calf serum and 2 ng/mL interleukin-3. All culture media were from GIBCO (Eggenstein, Germany).
Isolation of peripheral blood monocytes.
Buffy coats of healthy donors obtained from the blood bank were diluted
1:4 in phosphate-buffered saline and loaded on Histopaque 1177 (Sigma).
After centrifugation at 700g for 30 minutes at room temperature, the monocytic fraction was recovered and washed twice by
centrifugation, counted in a cell counter (Schärfe System, Reutlingen, Germany) before suspension in the adhesion buffer (RPMI
1640 containing 0.3% [wt/vol] BSA). Microscopic analysis was
performed to check cell viability and homogeneity before the experiments.
uPAR-transfected BAF-3 cells.
BAF-3 cells (an interleukin-3-dependent mouse pre-B-cell line) were
transfected by electroporation with uPAR cDNA (provided by Dr N. Behrendt, Copenhagen, Denmark) both in the sense and antisense
orientation in the expression vector pCDNA3. Cells were selected in the
presence of 0.8 mg/mL G418 and characterized for expression of uPAR by
flow cytometry, Northern blot analysis, and uPAR enzyme-linked
immunosorbent assay (ELISA) (data not shown).
Cell binding of radiolabeled VN.
Multimeric VN (100 µg) was labeled with 0.5 mCi of Na
125I (Amersham, Braunschweig, Germany) using Iodogen
(Pierce, Oud Beijerland, The Netherlands) according to a previously
outlined procedure.6 After separation on a Sephadex G-25
column (Pharmacia, Freiburg, Germany) suspended in Tris-buffered saline
containing 0.1% (wt/vol) BSA, the labeled protein was dialyzed against
the same buffer. The specific activity of VN in three different
preparations was 5 to 10 µCi/µg.
U937 cells were washed extensively with serum-free medium and
resuspended in Dulbecco's modified Eagle's medium (DMEM), pH 7.4, containing 25 mmol/L HEPES, 0.3% (wt/vol) BSA. 125I-VN
alone or together with excess cold ligand (at 50 µg/mL to measure
nonspecific binding) or other competitors was reacted for 2 hours at
4°C in a final volume of 0.15 mL. Aliquots of the binding mixtures
were layered on top of 0.2 mL DMEM containing 0.3% (wt/vol) BSA and
0.3 mol/L sucrose and centrifuged for 5 minutes at 12,000g. The
bottom tip of the tube containing the radioactive ligand-associated
cell pellet was counted in a  -counter.
Binding interactions in a purified system.
Polystyrene microtiter wells (high binding, type I; Costar,
Badhoevedorp, The Netherlands) were coated at a concentration of 5 µg/mL with purified soluble uPAR dissolved in 15 mmol/L
Na2CO3, 35 mmol/L NaHCO3, pH 9.6, and subsequently blocked with 3% (wt/vol) BSA. Binding of
125I-VN to the immobilized receptor was performed in a
final volume of 50 µL in the absence or presence of competitors as
indicated in the figure legends. Incubation was performed in
Tris-buffered saline, containing 0.05% (wt/vol) Tween 20, 0.3%
(wt/vol) BSA for 18 hours at 4°C, after which the wells were washed
and counted in a -counter. Nonspecific binding of
125I-VN to BSA-coated wells (in the absence of the
immobilized receptor) was used as an additional blank in all experiments.
Cell adhesion assays.
Cell adhesion to immobilized VN or FBG was performed according to
previously described protocols.6 Briefly, multiwell plates were coated with 2 µg/mL VN or 10 µg/mL FBG and blocked with 3% (wt/vol) BSA. BSA coated wells were used as controls to measure nonspecific adhesion. Differentiated U937 cells (24-hour preincubation with 100 nmol/L vitamin D3 and 2 ng/mL transforming growth
factor-) or BAF-3 cells were washed in serum-free medium and plated
onto VN- or FBG-coated wells for 60 to 90 minutes at 37°C in the
absence or presence of additives in serum-free medium containing 0.3% (wt/vol) BSA. In some experiments, HEPES-buffered saline (25 mol/L HEPES, pH 7.4, and 130 mmol/L NaCl) with or without Ca2+ (1 mmol/L) and Mg2+ (1 mmol/L) was used as the adhesion buffer
and the adhesion assay was limited to 30 minutes. In case of adhesion
to FBG, differentiated U937 cells were also incubated with 20 ng/mL
phorbol ester (PMA) as a positive control of 2-integrin
activation. Thereafter, the wells were washed and the number of
adherent cells was measured by quantifying the absorbance of crystal
violet staining at 590 nm.
Adhesion of myelomonocytic HL60 cells to cultured human umbilical vein
endothelial cells was performed according to a previously published
protocol.17 Briefly, differentiated HL60 cells were preincubated with different stimulators in the absence or presence of
MoAb 60.3 as outlined in the legend to Fig 6 and then allowed to adhere
to confluent endothelial cell monolayers. After incubation and washing,
adherent HL60 cells were quantified.
Zn2+ affinity chromatography.
Metal affinity chromatography resin (Pharmacia) was charged with
Zn2+ and used as described before for uPA
purification.34 Batch chromatography was performed with 5 µg each of multimeric VN, uPAR, and uPA. Proteins were bound in
phosphate-buffered saline and after washing they were eluted with the
same buffer containing 0.05 mol/L EDTA. The flow-through fraction and
the eluate were analyzed by denaturing polyacrylamide gel
electrophoresis in the presence of sodium dodecyl sulfate followed by
silver staining.
Flow cytometry.
HL60 cells (2.5 × 105) were washed twice with
HEPES-buffered saline and incubated in the absence or presence of 100 µmol/L Zn2+ or other test substances for 20 minutes at
room temperature. After washing, the cells were incubated with
saturating concentrations of mouse-anti-human MoAb 24 or MoAb 2LPM19c
for 30 minutes on ice. Cells were washed again, resuspended in HEPES
buffer and phycoerythrin-conjugated (Fab')2 fragment
of goat-anti-mouse IgG (Dianova, Hamburg, Germany) was added in
saturating concentrations for 30 minutes on ice. After washing and
resuspension, mean fluorescence of 10,000 cells was measured in a flow
cytometer (Becton Dickinson, Heidelberg, Germany). Nonspecific
fluorescence was determined using an isotype-matched mouse IgG as
primary antibody.
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RESULTS |
Zn2+-induced adhesion of U937 cells on VN.
Adhesion of myelomonocytic cells to VN is mediated via uPAR. uPA or its
isoforms containing the uPAR binding domain increase the affinity of
this interaction, thereby enhancing adhesion of monocytic cells to
VN.6,13 U937 or HL60 cells were differentiated for 24 hours
with vitamin D3 and transforming growth factor- along
the monocyte lineage, which results in a concomitant upregulation of
uPAR on these cells, as well as their adhesiveness to VN.
The effect of divalent cations Mn2+, Fe2+,
Co2+, Cu2+, Ni2+, Cd2+,
Hg2+, and Zn2+ in a concentration range between
5 and 100 µmol/L on adhesion of U937 cells to VN was tested. From
these divalent cations, only Zn2+ induced a substantial
increase in adhesion up to fivefold to sixfold, whereas
Mn2+ had a much smaller effect (Fig
1A). Zn2+ did not influence the
adhesion on a control substrate (BSA) and there was no toxicity of
these low concentrations of divalent cations used. The
adhesion-promoting effect of Zn2+ at 100 µmol/L almost
reached that of uPA at 50 nmol/L (Fig 1B). The adhesive effects of
Zn2+ and uPA were additive (not shown). Adhesion due to
Zn2+ and uPA on VN was inhibited by PAI-1 and MoAb R3
directed against domain 1 of uPAR, as well as by MoAb 13H1 against VN,
which blocks the interaction of VN with uPAR6 (Fig 1B),
whereas a control monoclonal antibody was ineffective.
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| Fig 1.
Adhesion of U937 cells on vitronectin in response to
divalent cations and uPA. (A) Differentiated U937 cells were allowed to
adhere on VN in response to Zn2+ ( ),
Mn2+ ( ), Co2+ ( ), Ni2+
( ), or Cu2+ ( ). The data are expressed as percent
of control as represented by adhesion of U937 cells in the absence of
any stimulus (mean ± SEM, n = 3). Similar results were obtained in
three separate experiments. (B) The adhesion of U937 on VN was
performed without stimulus ( ) or in the presence of 50 µmol/L
Zn2+ or 50 nmol/L uPA respectively. In parallel wells, no
other additives ( ) or 20 µg/mL of MoAb R3 against uPAR ( ), 10 µg/mL of MoAb 13H1 against VN ( ), or 100 nmol/L PAI-1 ( ) were
present. A control antibody (20 µg/mL) did not affect adhesion (not
shown). Data are the mean ± SEM (n = 3) of a typical experiment.
Similar results were obtained in three independent experiments.
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The cation-chelators, 1-10-phenanthroline or captopril, blocked the
effect of Zn2+ on cell adhesion, whereas uPA-induced
adhesion was only marginally affected (Fig
2A, data for captopril not shown).
Zn2+-induced adhesion was reversible: after the adhesion
experiment for 2 hours, addition of PAI-1 led to an immediate
dissociation of cells irrespective of Zn2+- or uPA-induced
adherence. 1-10-Phenanthroline induced a slower detachment of
Zn2+-stimulated, adherent cell, but did not affect the
cells that adhered in response to uPA (Fig 2B).
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| Fig 2.
Zn2+- and uPA-induced adhesion of U937
cells: effect of inhibitors. (A) Adhesion of U937 cells on VN-coated
wells in response to 50 µmol/L Zn2+ () or 50 nmol/L
uPA () was performed in the absence or presence of various
concentrations of 1-10-phenanthroline. Basal adhesion in the absence of
uPA or Zn2+ was 0.05 to 0.1 units of absorbance. Data
represent the mean ± SEM (n = 3) of a typical experiment. Similar
results were obtained in three separate experiments. (B) The adhesion
of U937 cells in response to 50 nmol/L uPA (filled symbols) or 50 µmol/L Zn2+ (open symbols) was performed for 2 hours.
Thereafter, 100 nmol/L PAI-1 (circles) or 500 µmol/L
1-10-phenanthroline (squares) was added for different time intervals as
indicated, and residual adherent cells were quantitated. Data represent
the mean ± SEM (n = 3) of a typical experiment. Similar results
were obtained in three separate experiments.
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Zn2+-induced adhesion of uPAR-transfected
BAF-3 cells.
To further define the specificity of the effects of Zn2+,
the adhesion on VN of BAF-3 cells (devoid of endogenous uPAR)
transfected with sense or antisense uPAR cDNA was investigated.
uPAR-expressing BAF-3 cells adhered to VN after stimulation by uPA or
Zn2+, and this effect could be attributed to uPAR as
evidenced by inhibition with MoAb R3 (Fig
3). Moreover, BAF-3 cells with
uPAR-antisense expression, either under control conditions or upon
stimulation with uPA or Zn2+, respectively, showed no
adherence to VN. This provides strong evidence that the
adhesion-promoting activity of Zn2+ is due to increasing
the strength of the VN-uPAR interaction.
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| Fig 3.
Adhesion of uPAR-transfected BAF-3 cells on VN. The
adhesion of BAF-3 cells transfected with uPAR-sense or uPAR-antisense
cDNA as indicated was performed in response to 50 µmol/L
Zn2+ or 50 nmol/L uPA, respectively, in the absence ()
or presence of 20 µg/mL of MoAb R3 against uPAR (). Data represent
the mean ± SEM (n = 3) of a typical experiment. Similar results
were obtained in three separate experiments.
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Influence of Zn2+ on the VN-uPAR
interaction.
The effect of Zn2+ on the VN-uPAR interaction was tested on
cells and in an in vitro system with isolated components. Like uPA, Zn2+ potentiated the direct binding of radiolabeled VN to
U937 cells, and the effects of Zn2+ and uPA were additive
(Fig 4A). Zn2+ increased
binding of 125I-VN to immobilized uPAR in a manner
comparable with uPA, whereas other cations were far less effective (not
shown). Again, the effects of Zn2+ and uPA on VN binding to
uPAR were additive. Both PAI-1 or MoAb R3 inhibited the effect of uPA
and Zn2+, while a control MoAb had no influence.
Zn2+ chelation with 1-10-phenanthroline abolished the
increase in 125I-VN binding to uPAR, whereas the increase
in binding mediated by uPA was not influenced (Fig 4B). These in vitro
results with isolated components indicated that Zn2+ has a
direct influence on VN-uPAR interaction by binding to these proteins
and changing their conformation. This possibility was confirmed by
testing the binding of these proteins to a Zn2+-Sepharose
column as has been previously demonstrated for uPA.34 Both
VN and uPAR are able to bind to the Zn2+-containing matrix
directly and could be eluted as intact proteins (Fig
5).
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| Fig 4.
Influence of Zn2+ on VN binding to uPAR.
(A) Binding of 125I-VN to U937 cells was performed in the
absence or presence of 50 µmol/L Zn2+ or 20 nmol/L uPA
alone or added together as indicated. Data (mean ± SEM, n = 3) are
expressed as specific binding (as determined by subtracting binding in
the presence of excess unlabeled VN). (B) Binding of
125I-VN to immobilized uPAR was performed in the absence or
presence of 50 µmol/L Zn2+ or 20 nmol/L uPA,
respectively. Either no additives () or 20 µg/mL MoAb R3 against
uPAR (), 500 µmol/L 1-10-phenanthroline (), or 100 nmol/L PAI-1
( ) were included as competitors. Data (mean ± SEM, n = 3) are
expressed as specific binding (represented as percent of control).
Similar results were obtained in at least three separate experiments.
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| Fig 5.
Binding of isolated proteins to Zn2+. After
metal-ion affinity columns were charged with excess Zn2+
or buffer alone, VN, uPAR, or single-chain urokinase type plasminogen
activator (ScuPA), respectively, were allowed to bind to
the column. Subsequent elution was performed with either binding buffer
alone (lanes 1, 3, 5) or containing 0.05 mol/L EDTA (lanes 2, 4, 6).
VN, uPAR, or ScuPA bound to the Zn2+ charged column, but
not to a control column (not shown). The respective authentic protein
bands are indicated by arrows on the right (monomeric and dimeric
VN).
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Zn2+-induced adhesion of monocytic cells on
FBG and endothelial cells.
Since 2-integrins have been reported to be activated by
divalent cations5,35,36 such as Mn2+ and
Zn2+, the possibility that Zn2+ increases
leukocytic cell adhesion on FBG was tested. Incubation of U937 cells
with physiologic concentrations of Zn2+ resulted in a
substantial induction of adhesion on FBG by threefold to fourfold (Fig
6) similar to Mn2+, whereas
other cations such as Fe2+, Co2+,
Cu2+, Ni2+, Cd2+, and
Hg2+ were far less effective (data not shown).
1-10-Phenanthroline inhibited the effect of Zn2+ without
influencing the PMA-induced adhesion (Fig 6A). The effect of
Zn2+ was comparable to that of PMA and was mainly mediated
by 2-integrins, since MoAb 60.3 against the human
2-subunit inhibited Zn2+-induced adhesion
(Fig 6B).
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| Fig 6.
Zn2+-induced adhesion of U937 cells on FBG.
(A) Adhesion of U937 cells on FBG-coated plates induced by 50 µmol/L
Zn2+ () or 20 ng/mL PMA () in the absence or
presence of various concentrations of 1-10-phenanthroline was measured.
Data represent the mean ± SEM (n = 3) of a typical
experiment. The basal adhesion in the absence of PMA or
Zn2+ was 0.05 to 0.1 absorbance units. Similar results
were obtained in three separate experiments. (B) The adhesion of U937
cells on FBG induced by 50 µmol/L Zn2+ or 20 ng/mL PMA
was measured in the absence () or presence of 10 µg/mL MoAb 60.3 against 2-integrin (). Data represent the mean ± SEM (n = 3) of a typical experiment. Similar results were
obtained in three separate experiments.
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As previously reported5 for Mn2+,
Zn2+ induced the activation of 2-integrins
by exposing an activation-dependent epitope on the
2-subunit without changing its surface expression on
HL60 cells as evidenced by flow cytometry (Fig
7A). Activation of
2-integrins also regulates the adhesion of leukocytes to
the endothelium, which takes place via the interaction between
2-integrins and counter-receptors such as ICAM-1 on
endothelial cells. In this system, Zn2+ promoted a fivefold
increase in HL60 cell adhesion to an endothelial monolayer, similarly
to the effect of Mn2+ or PMA (Fig 7B), while MoAb 60.3 inhibited cell adherence in all cases to baseline values indicative of
2-integrin dependency.
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| Fig 7.
Zn2+-dependent activation of
2-integrins and adhesion to endothelial cells. (A) Human
myelomonocytic HL60 cells were incubated in the absence or presence of
100 µmol/L Zn2+ for 20 minutes at room temperature, and
surface expression of an activation-dependent epitope (MoAb 24) on
2-subunit was quantitated by fluorescence-activated cell
sorter (FACS) analysis (filled curves). For comparison, quantitative
cell surface expression of 2-subunit was analyzed using
MoAb 2LPM19c, which recognizes an epitope irrespective of the
activation state of the integrin. Nonspecific fluorescence was
determined using an isotype-matched mouse-IgG (open curves). The figure
shows one of three representative experiments. (B) Differentiated HL60
cells, preincubated for 30 minutes in the absence or the presence of
100 µmol/L Zn2+, 0.5 mmol/L Mn2+, or 10 ng/mL PMA as indicated, were allowed to adhere to confluent endothelial
cell monolayers in the absence () or presence () of MoAb 60.3 against 2-integrin. Data represent the mean ± SD (n
= 3) of a typical experiment. Similar results were obtained in three
separate experiments.
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The 2-integrins have been shown to contain a divalent
cation binding site that binds both Mn2+ and
Zn2+, and their binding is negatively
influenced35,36 by Ca2+ and Mg2+.
Moreover, the activation state of 2-integrins is
strongly influenced by uPAR.14,16,17 BAF-3 cells, which
express 2-integrins37 transfected with uPAR
in the sense and antisense orientation, were tested for adhesion on
FBG. Zn2+, Mn2+, and PMA induced the adhesion
of uPAR transfected cells, but not that of control transfected cells in
normal adhesion buffer containing physiologic concentrations of
Ca2+ and Mg2+ (Fig
8A). This adhesion was inhibited by a
blocking MoAb for mouse 2-integrins, Game 46 (Fig 8A),
but not a control MoAb (data not shown). In a divalent cation-free
buffer, Mn2+could stimulate the adhesion of both uPAR sense
and antisense transfected cells to FBG, but Zn2+ could
activate the adhesion of sense transfected cells only (Fig 8B). This
indicates that Mn2+ can directly activate
2-integrins, whereas Zn2+ needs the
intermediate uPAR. This hypothesis is also supported by the fact that
the Mn2+ effect is strongly inhibited in the presence of
physiological Ca2+ and Mg2+, whereas these two
divalent cations have no influence on the effect of Zn2+
(Fig 8B). These findings strongly imply that Zn2+ binding
to uPAR regulates the activation status of 2-integrins in these model systems.
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| Fig 8.
Adhesion of uPAR-transfected BAF-3 cells on FBG. (A)
Adhesion of BAF-3 cells, transfected with uPAR in the sense or
antisense orientation, was tested on FBG in response to
Mn2+ (50 µmol/L), Zn2+ (50 µmol/L), or
PMA (20 ng/mL), respectively, in the absence () or presence of 10 µg/mL of MoAb Game 46 () against mouse 2-integrins.
(B) A similar adhesion assay was performed, except that either
HEPES-buffered saline without () or with Ca2+ and
Mg2+ () were used as the adhesion buffer instead of
RPMI 1640. Data represent the mean ± SEM (n = 3) of a typical
experiment. Similar results were obtained in three separate
experiments.
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Zn2+-induced adhesion of blood monocytes.
Monocytic cells were isolated from peripheral blood and their adhesion
to VN- or FBG-coated plates was investigated. Monocyte adhesion to VN
was induced twofold by 50 nmol/L uPA and more than 2.5-fold by 100 µmol/L Zn2+, whereas the effect of Mn2+ was
much smaller (Table 1). As demonstrated
above for myelomonocytic cell lines, the adhesion-stimulating effect of
Zn2+ on peripheral blood monocytes was also dose-dependent
(not shown). When FBG was used as a substrate, Zn2+
promoted monocyte adhesion similar to Mn2+ or PMA
(Table 1).
| |
DISCUSSION |
Cell adhesion to the endothelium and the provisional matrix, mainly
composed of VN or FBG/fibrin, is pivotal for leukocyte recruitment to
inflamed or injured tissue in order to initiate an adequate
inflammatory or wound healing response. These adhesive interactions
involve, largely, the uPAR system for VN and the 2-integrin-system for FBG/fibrin or the endothelial
monolayer as substrates.8,9,38 In the present work, we
demonstrate that physiologic concentration39 of the trace
element Zn2+ induces these different cell-matrix
interactions, as well as the firm adhesion of leukocytes to the
endothelium. Hence, Zn2+ could be a key regulator of
leukocyte adhesion in vivo.
In particular, Zn2+ enhanced the adhesion of leukocytic
cells on VN up to fivefold to sixfold, and this effect was specific for
Zn2+, as it could not be mimicked by other divalent cations
and was abolished by chelation with 1-10-phenanthroline or captopril. Zn2+-dependent adhesion engaged the uPAR system as deduced
from several lines of evidence: (1) a MoAb against uPAR blocked the
effect of Zn2+ on U937 cell adhesion; (2) Zn2+
induced the adhesion of uPAR-sense transfected BAF-3 cells, whereas BAF-3 cells transfected with uPAR-antisense did not respond; and (3) in
a purified system, Zn2+ promoted 125I-VN
binding to uPAR-expressing cells and to immobilized uPAR. The adhesion
promoting effect of Zn2+ in the VN-uPAR system was similar
to that of uPA, and both components were additive in their function. We
hypothesize that uPA and Zn2+ can induce a conformational
alteration in VN and/or uPAR, thus favoring their interaction. This is
also supported by our observation that both VN and uPAR can directly
bind to Zn2+. Elucidation of the Zn2+ binding
site(s) on uPAR and VN may explain how these two proteins interact in
order to mediate cell adhesion. In an analogous manner, Zn2+ increased the 2-integrin-mediated
adhesion of leukocytic cells on FBG/fibrin and endothelial cells, which
was inhibited by MoAb 60.3 or Game 46 against
2-integrins. The specificity of Zn2+ was
demonstrated by its ability to expose an activation-dependent epitope
on this integrin.
Although Zn2+ may stimulate monocytic cell adhesion to FBG
and the endothelium by direct activation of 2-integrins,
Zn2+-dependent engagement of uPAR appears to be crucial, as
deduced from our data. In a completely divalent cation-free buffer,
2-integrin-expressing BAF-3 cells without uPAR could
adhere strongly to FBG in the presence of Mn2+ (inhibited
by MoAb Game 46), but not in the presence of Zn2+. Hence,
Zn2+ binding to 2-integrins35,36
was not sufficient to stimulate adhesion to FBG. However, in
uPAR-transfected BAF-3 cells, Zn2+ induced a robust
increase in adhesion under the same divalent cation-free conditions,
indicating that Mn2+ activates 2-integrins
directly, while Zn2+ does so via uPAR activation.
Zn2+, Mn2+, Cu2+, Ca2+,
Mg2+, and Co2+ bind the I domain of the
-chain of 2-integrins with high
affinity.35,36 Ca2+ and Mg2+ can
inhibit Mn2+-stimulated 2-integrin-mediated
events based on direct competition at the divalent cation binding
site.35,36 Ca2+ and Mg2+ could
inhibit the adhesion of Mn2+ stimulated BAF-3 cells (with
or without uPAR) as expected, whereas Zn2+-induced adhesion
of uPAR transfected BAF-3 cells could not be influenced by
Ca2+ and Mg2+ at all, indicating that
Zn2+ does not activate adhesion by the same mechanism as
did Mn2+. Thus, Zn2+ induced
2-integrin-dependent adhesion to FBG or the endothelium through the activation of uPAR. We also observed some
Mn2+-induced cell adhesion to VN, reflecting a
"reverse" regulation of uPAR by 2-integrins,
corroborating earlier studies that showed uPAR-dependent cell
adhesion16 is influenced by activation of 2-integrins with Mn2+.
In a more complex physiologic environment, the cofactor role of
Zn2+ for other biologic systems such as the matrix
metalloproteinases should not be neglected. In a regulated manner,
these Zn2+-dependent endopeptidases efficiently degrade
matrix/tissue components required for cell migration and invasion and
for the release of stored growth factors.22,40 Furthermore,
the cell contact-dependent production and secretion of large quantities
of various matrix metalloproteinases by macrophages41 may
involve Zn2+as well. The administration of Zn2+
chelators such as captopril to inhibit matrix metalloproteinases was
used to prevent cancer cell invasion42 or tumor
angiogenesis43 in different models. Yet, it remains to be
clarified to what extent the presented functions of Zn2+ as
an inducer of cell adhesion are affected during therapy with cation chelators.
In addition to the plasma physiologic concentration of
Zn2+, platelets contain substantial
quantities44 of stored Zn2+ and release this
pool into regions of tissue damage, where Zn2+ may exert
its proadhesive and chemotactic effects for recruiting inflammatory
blood cells. Moreover, Zn2+ from implanted metal
biomaterials in the vasculature may also accumulate at the sites of
implantation.30 Conversely, in Zn2+ deficiency
states, there is a decline in the B- and T-cell-dependent immune
responses with increased susceptibility to bacterial, viral, and
parasitic challenges,45 and more relevant to the current study, there is a decline in neutrophil adhesion.46 These
causally linked observations, together with the data presented here,
suggest that Zn2+ may be a crucial regulator of leukocyte
adhesion and the extravasation of phagocytic cells into inflamed or
injured tissue.
| |
ACKNOWLEDGMENT |
The technical assistance of Barbara Yutzy, Uwe Schubert, and Thomas
Schmidt is greatly appreciated. We also acknowledge the generous gift
of reagents from Drs H.R. Lijnen (University of Leuven, Leuven,
Belgium), and G. Hoyer-Hansen and N. Behrendt (Finsen Laboratory,
Copenhagen, Denmark).
| |
FOOTNOTES |
Submitted July 28, 1998; accepted December 22, 1998.
Supported by Grant No. Pr 327/1-4 from the Deutsche
Forschungsgemeinschaft, Bonn, Germany, and the Novartis Foundation.
This work is part of the MD thesis of T.C. at the Department of
Medicine, Justus-Liebig-Universität Giessen, Germany.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Sandip M. Kanse, PhD, MPI,
Kerckhoff-Klinik, Sprudelhof 11, 61231 Bad Nauheim, Germany; e-mail:
sandip.kanse{at}kerckhoff.med.uni-giessen.de.
| |
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ABSTRACT
INTRODUCTION