|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1221-1230
Cooperative Activity of  4 1 and 47 Integrins in Mediating
Human B-Cell Lymphoma Adhesion and Chemotaxis on Fibronectin Through
Recognition of Multiple Synergizing Binding Sites Within the Central
Cell-Binding Domain
By
Zhinan Yin,
Emiliana Giacomello,
Elena Gabriele,
Luciano Zardi,
Shin-ichi Aota,
Kenneth M. Yamada,
Barbara Skerlavaji,
Roberto Doliana,
Alfonso Colombatti, and
Roberto Perris
From the Division for Experimental Oncology 2, Centro di Riferimento
Oncologico Aviano, Istituto Nazionale Centroeuropeo, Aviano, Italy; the
Department of Evolutionary and Functional Biology, University of Parma,
Parma, Italy; the Istituto Nazionale per la Ricerca sul Cancro, Centro
di Biotecnologie Avanzate, Genova, Italy; the Craniofacial
Developmental Biology and Regeneration Branch, National Institute of
Dental Research, National Institutes of Health, Bethesda, MD; and the
Dipartimento di Scienze e Tecnologie Biomediche, University of Udine,
Udine, Italy.
 |
ABSTRACT |
We have quantitated the relative contributions of the constitutively
active  4 1 and 47 integrins and the domains embodying their
cognate binding sites in mediating human B-cell lymphoma adhesion and
chemotaxis on fibronectin. By cooperating, the central cell-binding and
IIICS carboxy-terminal domains were entirely responsible for the
adhesion activity displayed by fibronectin, and their relative
contribution to this process was estimated to be 30% versus 70%.
Assessment of the leukocyte-substrate binding strength (ie, dynes/cell)
indicated a 10-fold higher avidity of the cell-IIICS domain
interaction. The two integrins interchangeably recognized both domains,
but differed quantitatively in their participation in the adhesive
event, as well as in domain preference. The use of 3Fn (according to
the nomenclature proposed by Bork and Koonin [Curr Opin Struct
Biol 6:366, 1996] for the type III fibronectin
modules) module-specific antibodies and recombinant polypeptides showed that 4 integrins recognized both the RGD sequence (3Fn10) and an apparently novel synergistic site located within the 3Fn8 module; even in this case, the integrins displayed a
distinct binding site preference. Interleukin-1
(IL-1)/IL-2-induced chemotaxis also involved cooperative function
of the central cell-binding and IIICS domains, but the mechanisms
regulating this phenomenon differed markedly from those controlling
cell adhesion. First, the relative contribution of the individual
domains was comparable, but neither of the individual domains promoted
migration to the extent observed on intact fibronectin. Secondly,
41 and 47 integrins were both involved in the
domain-binding necessary for initiation of migration, but the relative
contribution of each receptor in the chemotactic process was less
disparate than for initial cell adhesion. Thirdly, the mode by which
chemotactic B-lymphoma movement was supported by the central
cell-binding domain differed from that sustaining cell adhesion in that
it involved independent recognition of either the 3Fn8 or the 3Fn9 module, which acted in synergy with the 3Fn10 module. Our data provide
novel evidence concerning the relative importance of the constitutively
active 41 and 47 integrins for the interaction of B-cell
lymphoma cells with fibronectin, and they emphasize a multiple and
diverse recognition of sites responsible for either anchorage or
locomotion of tumor leukocytes on this matrix molecule.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE INTERACTION OF normal and neoplastic
leukocytes with fibronectin is thought to be critically involved in the
regulation of leukocyte trafficking through tissues. The central
cell-binding domain of fibronectin contains the RGD sequence, which is
believed to be the primary cell adhesion site of this domain, and at
least two additional, spatially well-separated cell-binding sites that are thought to operate synergistically.1-4 The
carboxy-terminal IIICS variable region also contains two potential
cell-binding sites designated CS-1 and CS-5 (connecting segment 1 and
5).5-8 We and other investigators have recently
demonstrated that, although the RGD sequence is a critical determinant
for the  51-mediated adhesion of neoplastic leukocytes to
fibronectin, optimal interaction of the cells requires recognition of a
cooperative cell adhesion site residing within 3Fn9 (according to the
nomenclature proposed by Bork and Koonin9 for the type III
fibronectin modules) and defined by PHSRN.8,10
The arginine residue is the key amino acid of this site11
and 51 integrin binding to it seems to be strongly influenced by
the activity state of the receptor8 as well as the proper
spatial orientation of the 3Fn9-10 ligand modules.12
The 41 and 47 integrins have been described as
characteristic lymphocyte integrins reacting with the CS-1 cell
attachment site within the IIICS region,7,13,14 in addition
to a series of other cell surface and extracellular ligands, including
vascular cell adhesion molecule-1 (VCAM-1), mucosal
addressin cell adhesion molecule-1 (MadCAM-1), invasin,
thrombospondin, and von Willebrand factor propeptide. However, several
points remain unclear concerning this apparently rather broad
interaction repertoire. First, it is not yet unambiguously determined
whether all these ligands are truly functional in vivo. Secondly, it is
not clear whether both integrins recognize all putative ligands equally
well, simply by virtue of having in common the 4 subunit. Thirdly,
the relative roles of the integrins in mediating adhesion and migration
of lymphocytes on fibronectin remain to be firmly established.
B-lymphoid cells, largely lacking 51, may alternatively use the
41 integrin to bind to the central cell-binding domain of
fibronectin,15,16 but this putative RGD-recognition seems
to be under strict control of the activation state of the receptor.
This is believed because only integrin complexes in which the 1
subunit is artificially activated by ligation with the activating
antibody TS2/16 are capable of exhibiting this binding
specificity.15,16 Finally, although the
41 integrin may recognize multiple binding sites within the
fibronectin molecule, the present evidence is that the human 47
integrin binds to the CS-1 site and a putative recognition
sequence recently identified within 3Fn5.17-19
We have previously shown that two highly malignant cell lines, Ci-1
(lymphoblastic B-cell lymphoma) and Sc-1 (noncleaved Burkitt's type
lymphoma), bind constitutively to fibronectin and express high levels
of 4 integrins and low to poorly detectable levels of
51.20 In this study, we have used these model cells
to determine the relative contribution of the 41 and 47
integrins in the processes of cell attachment and cytokine-induced
chemotaxis in response to the central versus IIICS cell-binding
domains. Thus, we have addressed comparatively both the relative
contributions of the two primary fibronectin domains in
adhesion/migration phenomena and of the sites within these domains that
are recognized by the two integrins.
| |
MATERIALS AND METHODS |
Antibodies and their specificities.
The specificity and sources of the monoclonal antibodies (MoAbs) used
in this study were as follows: anti-1 MoAb 4B4 was purchased from
Coulter Scientifics, Ltd (Palo Alto, CA); activating anti-1 antibody
TS2/16 was provided by Dr Martin Hemler (Dana-Farber Cancer Research
Institute, Harvard Medical School, Boston, MA); anti-4 MoAb HP2-1
was kindly provided by Dr Francisco Sanchez-Madrid (Servicios de
Immunologia, Hospital de la Princesa Università Autònoma de
Madrid, Madrid, Spain); anti-4 MoAb P4G2, anti-5 MoAb P1D6, and
anti-51 complex MoAb JBS5 were purchased from Chemicon
International (Temecula, CA); anti-47 MoAb Act-1 was kindly
provided by Dr Andrew Lazarovitz (Department of Nephrology, University
of Ontario, London, Ontario, Canada); and anti-4 MoAb PS2
(hybridoma) was purchased from ATCC (Rockville, MD).
Antibodies against fibronectin included MoAb 333 directed against an
epitope localized within the Fn3-10-11 module of
fibronectin4; MoAb HFN-7 (which was purified from the
corresponding hybridoma cells obtained from the ATCC) is directed
against an epitope within the 3Fn9 module10; and MoAb IST-6
recognizes the 3Fn8 module.10 Antibody P3D4 (Chemicon
International) was raised against a carboxy-terminal fragment of
fibronectin and is known to block the interaction of cells with the
38-kD and 58-kD fragments of the A and B chains of
fibronectin.21 MoAb FN15 (Sigma, St Louis,
MO) reacts with the central cell-binding domain of
fibronectin, but does not affect cell adhesion.
Fibronectin fragments, recombinant polypeptides, and synthetic
peptides.
Bacterial expression proteins of molecular weight
(Mr) 110,000 (110 kD) and Mr
120,000 (120 kD), the latter one including the EDIIIB
alternatively spliced 3Fn module, correspond to the entire central
cell-binding domain of fibronectin and were produced essentially
according to the procedures described in Moyano et al.19
These recombinant proteins were indistinguishable in their attachment-
and migration-promoting activity, indicating that the
EDIIIB module did not contribute to the cell biological
activities of the protein in our system. We have therefore used these
proteins interchangeably throughout the study, and for simplicity, we
consistently refer to the 110-kD polypeptide when describing the data
related to these polypeptides. The Mr 38,000 (38 kD)
tryptic fragment of fibronectin, comprising the Hep II domain and the
putative cell attachment sites CS-1 through CS-3 of the IIICS region,
was a generous gift from Dr Angeles Garcia-Pardo (Department of
Immunology, Centro de Investigationes Biologicas, Madrid, Spain). A
recombinant protein of Mr 52,600 and encompassing the
carboxy-terminal 3Fn12-15 modules and the complete IIICS domain (ie,
CS-1 through CS-5)22 was kindly provided by Dr Martin
Humphries (Department of Biochemistry and Molecular Biology, University
of Manchester, Manchester, UK). Because the two carboxy-terminal
fragments supported cell adhesion to equivalent extents, they were used
interchangeably throughout the study and described as the 38-kD
fragment. Synthetic peptides with the composition RGDNP,
GPenGRGDSPCA, and GDRGDSPASSK were purchased from
GIBCO BRL, Life Technologies (Paisley, UK), and peptides with the
composition GRGDSP, GRGESP,
DELPQLVTLPHNLHGPEILDVPST (CS-1), GEEIQIGHIPREDVDYHLYP (CS-5),
and DRVPHSRNSIT (B peptide) were synthesized on an automated
Milligen 9050 peptide synthesizer (Millipore, Bedford, MA) using Fmoc
chemistry and were purified by reverse-phase high-performance liquid
chromatography (HPLC) according to standard procedures
and analyzed for purity by amino acid analysis (identified active
recognition sequences of these peptides are underlined).
GRGD-containing peptides were found to be equally active and were used
interchangeably throughout the study. Recombinant bacterial
polypeptides corresponding to the fibronectin modules 3Fn4-6, 3Fn7-9,
3Fn9, and 3Fn10 were produced according to the procedures described in
Moyano et al.19 These polypeptides are arbitrarily denoted
R-3Fn4-6, R-3Fn7-9, R-Fn9, and R-Fn10. Another series of recombinant
polypeptides was produced from previously generated cDNA clones by
reamplification with adapter primers and cloning into the pQE-30 high
expression vector (Qiagen, Hilden, Germany), in which the recombinant
protein is fused to an N-terminal 6-His affinity tag. The positive
clones were sequenced to rule out Taq polymerase errors, and the
recombinant fragments were expressed by standard protocols. To improve
refolding of these recombinant polypeptides, the fusion products were
bound to a Ni2+ chromatography matrix, urea was removed,
and the bound proteins were eluted with phosphate-buffered saline
(PBS). The purity of the final products was ascertained by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This
protocol was used to generate the following polypeptides.
R-3Fn3/8R (where R refers to the critical arginine of the
recognition PHSRN motif) encompassing the 3Fn8 and 3Fn10 modules, but
lacking the 3Fn9 module and including the PHSRN sequence derived from
the 3Fn9 module, was generated. This sequence was inserted in the most
homologous region identified between the 3Fn8 and 3Fn9
modules.23 R-3Fn8/10D is identical to
R-3Fn8/10R, with the exception that the arginine residue in
PHSRN was substituted by an aspartic acid residue (ie,
PHSDN).23 R-3Fn6-10SPSDN encompasses 3Fn6-10,
but carries a 16-amino acid mutation including a substitution of PHSRN
 SPSDN of the 3Fn9 synergy sequence.11
Cell adhesion assay (centrifugal assay for fluorescence-based cell
adhesion [CAFCA]).
The cell adhesion assay CAFCA (TECAN Group)23,24 used in
this study is based on differential
centrifugation25 and represents an extensive
refinement of an assay previously described by our group.10,20 Major innovations include the use of
commercially available six-well strips of flexible polyvinyl chloride
(PVC) denoted CAFCA miniplates (TECAN Austria, Salzburg,
Austria), covered with double-sided tape (bottom units),
and fluorescent cell labeling with the vital fluorochrome calcein AM
(Molecular Probes, Inc, Eugene, OR) for 20 minutes at 37°C. Labeled
cells were rinsed extensively in Ca2+- and
Mg2+-free PBS followed by one rinse in 50 mmol/L Tris-HCl,
pH 7.2, containing 150 mmol/L NaCl (TBS) and 1 mmol/L EDTA, and then
aliquoted into the bottom CAFCA miniplates at a density of 1 to 3 × 104 cells/well. Cell adhesion to substrates was
assayed in TBS containing 0.1% bovine serum albumin (BSA), 1 mmol/L
MgCl2, and 2% India ink as a fluorescence quencher. In
some instances, cells were preincubated with the anti-integrin
antibodies (1 to 3 µg/106 cells) anti-1 (MoAb 4B4;
inhibitory), anti-1 (MoAb TS2/16; stimulatory), anti-4 (MoAb
HP2-1), anti-47 (MoAb Act-1), or anti-5 (MoAb P1D6) or with 1 to 100 µg of recombinant R-3Fn fragments in PBS for 20 minutes at
37°C and then plated onto the substrates. Synthetic peptides were
added directly to the cell adhesion medium at final concentrations of
0.01 to 1 mg/mL. CAFCA miniplates were placed on specifically devised
hard plastic holders (TECAN Austria) and centrifuged at 142g
for 5 minutes at 37°C to synchronize the contact of the cells with
the substrate. The miniplates were then incubated for 30 minutes at
37°C and subsequently mounted together with a similar CAFCA
miniplate lacking double-sided tape (top unit) to create communicating
chambers for subsequent reverse centrifugation. Tests were initially
performed to determine the minimal reverse centrifugal force required
to detach cells nonspecifically adhering to a BSA-coated substrate, but
yet sufficiently low to detect low-avidity binding to a matrix
substrate. This force was established to be 12g when applied
for 5 minutes at 37°C for the human B-cell lymphoma lines examined
herein. Other sets of experiments, in which the miniplate assemblies
were centrifuged at 12g, 45g, 100g,
177g, 399g, and 710g, were also performed to
determine the relative strength of cell adhesion to the substrate.
Higher centrifugation forces could not be used because they caused
detectable damage to the cells. The relative number of cells bound to
the substrate (ie, remaining bound to the bottom miniplates) and of
unbound cells (in wells of the top miniplates) was estimated by
top/bottom fluorescence detection in a computer-interfaced
SPECTRAFluor microplate fluorometer (TECAN Austria).
Fluorescence values were analyzed with the CAFCA software (TECAN
Austria) to determine the percentage of adherent cells of the total
cell population analyzed according to a previously devised
formula.24-26 Relative strengths of cell adhesion in
dynes/cell (Afd) were calculated as previously described24-26 according to the formula:
Afd = (Dc  Dm) × Vc × Fc, where Dc is the specific
density of the cell, previously established to be 1.07 g/mL25,26; Dm is the specific density
of the medium equal to 1.00 g/mL; Vc is the volume
of the cell; and Fc is the centrifugal force exerted on the
bound cell. The average cell diameter of Sc-1 and Ci-1 cells was
comparable and was established to be 9 µm. If the substrate contact
area of cells binding to a specific ligand is estimated,
Afd may also be converted to the recently proposed
adhesion constant  , ie, force-length2 (dynes/cm2).27 Statistical significance, as
determined using the Student's t-test, was set at P < .001.
Cell motility assays.
Transmigration experiments involving chemotactic movement of the cells
through a porous membrane were performed according to a novel
fluorescence-based motility assay denoted fluorescence-assisted transmigration invasion and motility assay (FATIMA; TECAN
Austria).26 Specifically devised Unicell-24 plates
containing Transwell-like units in which a fluorescence-shielding
polycarbonate membrane (Polyfiltronics, Inc, Boston, MA; TECAN Austria)
with 5-µm pores was mounted were coated with 5 to 20 µg/mL of
intact fibronectin or fibronectin-derived polypeptides (control wells
had uncoated membranes) as described for CAFCA (50 µL/well), followed
in some cases by incubation with various antifibronectin antibodies and extensive washing with RPMI. At the time of cell plating, the wells of
the lower tray of the Unicell-24 plates were filled with 600 µL of
RPMI with or without 5 ng/mL interleukin-1 (IL-1) or 50 ng/mL
IL-2 (ImmunoKontakt, Frankfurt, Germany), and 2 × 105
cells suspended in 100 µL of RPMI were added to the top of each transwell. Optimal cytokine concentrations were determined in separate tests.
In perturbation experiments with anti-integrin antibodies, cells were
preincubated for 30 minutes at 37°C with the various antibodies
before being aliquoted into the Unicell-24 plates. Pilot tests
indicated that a minimum of 24 hours of incubation was needed to
observe substantial transmigration; consequently, all of the
measurements were performed at 24 to 48 hours. Transwell units
containing cells that had not transmigrated were removed, and the
bottom trays containing the transmigrated cells were gently centrifuged
for 5 minutes at 8g. Subsequently, 20 µmol/L calcein AM was
added, and cells were further incubated for 20 to 30 minutes at
37°C, followed by fluorescence detection with the
SPECTRAFluor fluorometer. The numbers of cells transmigrated
were derived from the fluorescence values by extrapolation from
parallel dilution curves of labeled cells. The percentage of
transmigrated cells varied from 22% to 26% of the total added to the
system, and nonspecific passage through membranes in the absence of
surface coating was consistently less than 30% of the total migration
observed. Statistical significance was determined as for CAFCA.
| |
RESULTS |
Constitutive B-cell lymphoma interaction with the central and IIICS
fibronectin cell-binding domains.
Sc-1 and Ci-1 B-lymphoma cells constitutively expressing high levels of
41 and 47 (as confirmed by flow cytometry; data not shown)
adhered to both the carboxy-terminal, IIICS-containing 38-kD and to the
110-kD polypeptide corresponding to the entire central cell-binding
domain (Fig 1A). Stable attachment to 38 kD
and 110 kD did not require any prior chemical or immunological activation of the cells; accordingly, preincubation of the cells with
the 1-integrin activating antibody TS2/16 did not alter the adhesion
pattern of the cells (Fig 1A). Cell adhesion to both domains, as well
as to intact fibronectin, was entirely cation-dependent and was equally
promoted by physiological concentrations of either Mg2+ or
Mn2+ (1 to 3 mmol/L or 30 to 100 µmol/L, respectively).
However, Ca2+ failed to stimulate cell adhesion, even when
used at concentrations up to 10 mmol/L (not shown). The simultaneous
presence of Mg2+ and Mn2+ did not potentiate
cell binding, and the addition of Ca2+ to cells binding in
the presence of the former ions similarly did not affect the extent of
cell adhesion (not shown). Cell adhesion to the 38-kD fragment largely
paralleled that seen on intact fibronectin, when compared in molar
equivalents (Fig 1A) and corrected for the independently established
relative coating efficiencies of the proteins. The 110-kD and 120-kD
fragments also supported substantial cell attachment that, in molar
equivalents, corresponded to up to 30% of the maximal attachment seen
on intact fibronectin (Fig 1A). These results indicate that the
relative contributions of the two cell-binding domains to the promotion
of Sc-1/Ci-1 cell adhesion to fibronectin were 70% versus 30%.
View larger version (45K):
[in this window]
[in a new window]
| Fig 1.
(A) Sc-1 and Ci-1 cell adhesion to 38-kD fragment and
110-kD polypeptide with and without addition of the anti-1
activating antibody TS2/16. Data are presented as the ratio of cells
bound to each of the two proteins when compared with cells adhering to
the intact fibronectin molecule, after subtraction of nonspecific
binding to BSA (5% to 10%). The extent of cell attachment was
analyzed at various concentrations of the fragment/polypeptide and is
expressed as molar equivalents of intact fibronectin. (B) Relative
avidity of Sc-1 and Ci-1 cell binding to the 38-kD fragment and 110-kD
polypeptide assessed by applying increasing centrifugal forces to
detach the adherent cells.
|
|
Although fewer cells adhered to the 110-kD polypeptide than to the
38-kD fragment, the application of CAFCA using differential centrifugation forces to determine cell-substratum binding
avidities10,20,24-26 showed that Sc-1 cells attaching to
the two fragments did so with roughly equal avidity (~2.5 × 10 4 dynes/cell; Fig 1B). This avidity was comparable
to that detected on intact fibronectin. Ci-1 cells, on the other hand,
differed in the force of attachment to the two fragments, binding with estimated forces of 1.4 × 104 dynes/cell to
the 38-kD fragment and 2.0 × 105 dynes/cell to
the 110-kD polypeptide.
Comparative assays with a number of well-known B and T neoplastic
leukocytes expressing one, two, or all three of the integrins 51,
41, and 47 clearly demonstrated that cells expressing high
levels of 51 or medium/high levels of 41 (ie, Karpas 299, HUT78, and HUT102) strongly bound to the 110-kD polypeptide, whereas
Ramos cells exclusively expressing 4-integrins bound solely to the
38-kD fragment (Fig 2). Jurkat cells
expressing all three receptors at high levels, ie, 51, 41,
and 47, characteristically showed more extensive binding to the
110-kD polypeptide than to the 38-kD fragment, and this behavior
markedly differed from that of Sc-1 and Ci-1 cells (Fig 2). The
observation that Ramos cells failed to recognize the central
cell-binding domain in a constitutive manner28 is in
agreement with previous studies showing that this ability is conferred
to these cells only after immuno-mediated 1 integrin subunit
activation.15,16
View larger version (43K):
[in this window]
[in a new window]
| Fig 2.
Comparison between the constitutive attachment profiles
of Sc-1/Ci-1 cells and various neoplastic human T and B leukocytes
adhering to intact fibronectin, the 110-kD polypeptide, or the 38-kD
fragment. Integrin expression as determined by FACS was as follows:
Karpas, HUT78 and HUT102
51high/41low; Ramos
41high; and Jurkat
51high/41high/4/7high.
Each substrate molecule was coated at a concentration of 10 µg/mL
(not in molar equivalents). In all cases, binding to the control BSA
substrates was less than 10%.
|
|
Identification of the sites responsible for the tethering of Sc-1 and
Ci-1 cells to fibronectin.
A synthetic peptide containing the RGD cell-recognition motif did not
affect Sc-1 and Ci-1 cell attachment to either the 110-kD polypeptide
or 38-kD fragment, whereas it entirely blocked 51-mediated K562
(reference 51-positive/RGD-dependent cells) cell adhesion to the
110-kD polypeptide (Fig 3). Peptides
containing the previously identified synergistic sequence PHSRN (B
peptide) of the 3Fn9 module, and peptides corresponding to the
penultimate segment (CS-5) of the IIICS domain, similarly failed to
affect B-cell attachment to both the 110-kD polypeptide and 38-kD
fragment (Fig 3). In contrast, the CS-1 peptide substantially blocked
cell adhesion to both proteins (Fig 3). Preincubation of fibronectin
substrates with the function-blocking antibodies 333 (occluding the RGD
site) or P3D4 (blocking cell interaction with the CS-1 site) did not alter Sc-1 and Ci-1 cell adhesion to intact fibronectin when used alone
(Fig 4). In contrast, these antibodies
entirely abrogated cell binding when added in combination (not shown),
confirming that, at least in this system, these two domains accounted
for all cell adhesion activity exerted by the fibronectin molecule. Preincubation of 110-kD substrates with MoAb 333 resulted in a partial
inhibition of cell adhesion (55%), as did similar preincubations with
MoAb IST-6 directed against the 3Fn8 module. The scenario was different
for MoAb HFN-7 reacting with the 3Fn9 module, which failed to perturb
cell adhesion (not shown). The combination of MoAbs 333 and IST-6
entirely blocked cell binding to the 110-kD fragment, whereas the
combination of MoAbs 333 and HFN-7 did not yield an additive effect
(Fig 4). This latter finding indicated that abrogation of cell binding
by the combination of MoAbs 333 and IST-6 was a consequence of the
blockade of cooperating binding sites in 3Fn8 and 3Fn10 or possibly a
site residing within the overlapping region between 3Fn8 and 3Fn9 in
addition to the RGD site of 3Fn10.
View larger version (43K):
[in this window]
[in a new window]
| Fig 3.
Effects of synthetic peptides corresponding to known
cell-binding sites on cell adhesion to the 110-kD polypeptide and 38-kD
fragments. K562 cells, expressing constitutively high levels of
51 integrin, were used as reference cells to confirm the
inhibitory activity of RGD-containing peptides and to determine the
optimal blocking concentration. The optimum for these cells was
established to be 50 µg/mL of GRGDSP peptides, and raising the
concentration to 550 µg/mL still did not inhibit Sc-1/Ci-1 cell
binding. Dose-dependent inhibition curves with CS-1 peptides showed a
concentration optimum of 150 µg/mL peptide. B peptide and CS-5 were
tested at concentrations up to 850 µg/mL.
|
|
View larger version (50K):
[in this window]
[in a new window]
| Fig 4.
Inhibitory effects of antifibronectin antibodies on Sc-1
and Ci-1 cell adhesion to intact fibronectin and the 110-kD
polypeptide. Antibody P3D4 was used as a control antibody. Antibodies
were used at concentrations of 1 to 10 µg/mL.
|
|
Sc-1/Ci-1 cell adhesion to fibronectin is cooperatively mediated by
41 and 4<giGb7
integrins with diverse cell-binding site preferences.
The interaction of the Sc-1 and Ci-1 cells with intact fibronectin was
unaffected by anti-5 integrin subunit antibodies (MoAb P1D6;
Fig 5A and B) and an antibody against the
51 complex (MoAb JBS5; data not shown). In contrast, adhesion of
the cells to the intact molecule was entirely abrogated by MoAb HP2-1
against the 4 integrin subunit (Fig 5A and B). When added alone, the
anti-1 MoAb 4B4 partially blocked cell adhesion to intact
fibronectin, and the anti-47 MoAb Act-1 only had a marginal, but
significant, effect. Simultaneous addition of the two antibodies
strongly reduced the ability of cells to tether to fibronectin (Fig 5A
and B). Cell binding to both the 110-kD polypeptide and 38-kD fragment was also entirely abrogated after the addition of MoAb HP2-1 (Fig 5A
and B). However, cell adhesion to the 110-kD polypeptide was altered to
an equivalent extent by MoAbs 4B4 and Act-1 (Fig 5A and B).
View larger version (27K):
[in this window]
[in a new window]
| Fig 5.
Inhibitory effects of anti-integrin antibodies, used
alone or in combination, on Sc-1 (A) and Ci-1 (B) cell adhesion to
intact fibronectin and its cell-binding domains: P1D6, anti-5
(identical results were obtained with the anti-51 MoAb JBS5);
HP2-1, anti-4; 4B4, anti-1; and Act-1, anti-47. To
determine the optimal inhibitory concentration of each antibody,
antibodies were individually titrated in independent tests and found to
be optimally active at 1 to 3 µg/106 cells. (C) Relative
strength of substratum adhesion estimated for Sc-1 cells binding to the
38-kD fragment or 110-kD polypeptide in the presence or absence of
either MoAb 4B4 or MoAb Act-1, as determined by exposing bound cells to
the centrifugal forces indicated on the abscissa.
|
|
Exposure of Sc-1 cells that had attached to either the 38-kD fragment
or 110-kD polypeptide to progressively incremented centrifugal forces
in the alternative presence of MoAb 4B4 or Act-1 demonstrated that
binding to the 110-kD polypeptide was strongly dependent on the
activity of 47, whereas the opposite was true for cell attachment
to the 38-kD fragment (Fig 5C). Assessment of the relative adhesive
forces responsible for the cell-substratum interaction in the presence
of the antibodies yielded an Adf of 5.35 × 105 dynes/cell for cells attached to the 38-kD
fragment in the presence of MoAb Act-1 versus an
Adf of 1.40 × 105
dynes/cell in the presence of MoAb 4B4, when extrapolated to a
centrifugal force retaining 50% of the cells originally bound. On the
other hand, an Adf of 2.25 × 105 dynes/cell versus an Adf of
1.20 × 105 dynes/cell was estimated for cells
attaching to the 110-kD polypeptide in the presence of MoAbs 4B4 and
Act-1, respectively.
Localization of the sites responsible for the
41- and
47-mediated adhesion of Sc-1 cells to
the central cell-binding domain of fibronectin.
All R-3Fn polypeptides composed of the 3Fn8, the 3Fn10, or both modules
strongly inhibited Sc-1 cell binding to the 110-kD polypeptide
(Fig 6A), whereas polypeptides R-3Fn9 and
R-3Fn4-6 (not shown) were completely inactive. A marked difference was also observed between the inhibitory efficiency of polypeptides R-3Fn6-10SPSDN and R-3Fn8/10D and the other
polypeptides, with the former displaying competing activity comparable
to the larger 110-kD polypeptide (Fig 6A). Sc-1 cell attachment to the
immobilized recombinant polypeptides R-3Fn7-9, R-3Fn9-10, and R-3Fn10
was significantly (38% to 61%; P < .001) less pronounced
than that to the intact 110-kD fragment (even when the polypeptides
were coated onto plastic at a >10-fold molar excess in comparison
with the parent fragment), whereas fragments R-3Fn9 and R-3Fn4-6 were
completely inactive (Fig 6B). On the other hand, cell attachment to
polypeptide R-3Fn8/10D was not significantly
distinguishable (P > .01) from that seen on the 110-kD
fragment when assayed at a 1:5 molar ratio (Fig 6B). However, in
contrast to the findings with immobilized polypeptides, the inhibitory
effect of soluble polypeptides R-3Fn6-10SPSDN and
R-3Fn8/10D was not distinguishable from that of
polypeptides R-3Fn7-9, R-3Fn9-10, and R-3Fn10 (Fig 6A). This
observation indicated that, in liquid phase, the putative cell
recognition sites exposed in both the R-3Fn8/10D and
R-3Fn10 polypeptides were of equal importance for the anchorage of
cells to the central cell-binding domain.
View larger version (36K):
[in this window]
[in a new window]
| Fig 6.
(A) Sc-1 cell binding to the 110-kD polypeptide after the
addition of 110-kD or R-3Fn polypeptides. The dotted line indicates
cell adhesion in the absence of competing polypeptide. Inhibition
resulting from 110 kD, R-3Fn7-9, R-3Fn9-10, R-3Fn10,
R-3Fn6-10SPSDN, and R-3Fn8/10D are
significantly different. (B) Sc-1 cell adhesion to immobilized
recombinant polypeptides in the presence or absence of anti-integrin
antibodies at their optimal blocking concentrations. The amount of
R-3Fn polypeptide coated onto plastic, or added in solution, was not in
molar equivalents, because it is well known that the biological
activity of such fragments is rather low3,11,23 and must be
compensated for by using an excess of added protein. Fragments were
therefore used at concentrations up to 100 µg/mL, based on
independent dose-dependency tests, which established that the fragments
were not significantly more active above the indicated concentrations.
Antibody inhibition assays reported here do not include immobilized 110 kD, R-3Fn9, or R-3Fn4-6.
|
|
In accordance with data obtained using the larger 110-kD polypeptide,
cell attachment to polypeptides R-3Fn7-9, R-3Fn9-10, R-3Fn10, and
R-3Fn8/10D was completely blocked by the anti-4 MoAb
HP2-1 (Fig 6B). In the case of the R-3Fn9-10 and R-3Fn10 polypeptides,
Sc-1 cell adhesion was also completely blocked by the anti-1 MoAb
4B4, whereas cell attachment to the R-3Fn7-9 and R-3Fn8/10D
polypeptides was either unaffected or only partially (45%) affected (Fig 6B). The anti-47 MoAb Act-1, on the other hand, eliminated entirely cell binding to R-3Fn7-9 and reduced cell adhesion to the
R-3Fn8/10D polypeptide by 75% (Fig 6B).
Differential role of the central cell-binding and IIICS fibronectin
domains in cytokine-induced chemotaxis.
Both IL-1 and IL-2 independently stimulated the chemotactic movement
of Sc-1 cells in response to the 110-kD and 38-kD substrates, but in
contrast to what was observed for cell adhesion, these proteins did so
to a largely comparable extent. However, none of the individual
molecules was capable of promoting migration to an extent similar to
that observed with intact fibronectin (Fig
7A). Thus, assuming a comparable protein coating efficiency of the
polycarbonate membranes as for plastic, the results indicated that the
motility-promoting activity of fibronectin induced by the
chemoattractive stimuli involved a 50% relative contribution of each
of the central cell-binding and IIICS domains, and optimal leukocyte
movement was only attained when both domains exerted their promoting
effects in a cooperative fashion.
View larger version (32K):
[in this window]
[in a new window]
| Fig 7.
(A) Dose-related motility response of Sc-1 cells to
intact fibronectin, 110-kD polypeptide, or 38-kD fragment under the
chemoattractive stimuli of IL-1/IL-2, as monitored FATIMA (see
Materials and Methods). (B) IL-1/IL-2-elicited chemotaxis of Sc-1
cells on intact fibronectin (10 µg/mL), 120-kD polypeptide, or 38-kD
fragment (20 µg/mL) in the presence of antibodies to 4 (HP2-1 and
PS2), 1 (4B4), or 47 (Act-1) integrins, added at their maximal
inhibitory concentration. (C) Effects of antibodies against the central
cell-binding domain of fibronectin on IL-1/IL-2-induced Sc-1 cell
chemotaxis. The membrane to be transmigrated was coated with the 110-kD
polypeptide (20 µg/mL) followed by incubation with the different
antibodies as described for CAFCA. FN15 corresponds to a cell-binding
domain-directed MoAb with no reported functional effects.
|
|
The IL-1/IL-2-induced chemotaxis of Sc-1 cells, both in response to
intact fibronectin and to the isolated 110-kD and 38-kD polypeptide/fragment, was in all cases effectively blocked by MoAb 4B4,
HP2-1, or Act-1. As expected, the antimurine 4 integrin antibody
PS2, known to display only a marginal cross-reactivity with the human
integrin homologue, had only a minor effect (Fig 7B). The substantially
less effective inhibition caused by addition of the anti-47 MoAb
Act-1 on cell movement on the 38-kD fragment, when compared with the
110-kD polypeptide and intact fibronectin (Fig 7B), was largely
consistent with its effect on initial cell adhesion. However, in
contrast to cell adhesion, estimation of the relative contribution of
the two integrin receptors indicated 40% versus 60% in the
contribution of the 47 and 41, respectively, in the binding
to the central cell-binding domain. None of the recombinant R-3Fn
polypeptides supporting cell adhesion was able to promote chemotactic
migration of Sc-1 cells significantly above background levels (ie, on
uncoated membranes; data not shown). Yet another difference between the
requirements for initial cell adhesion and chemotaxis was that the
single MoAbs 333 and IST-6 significantly blocked the chemotactic
movement elicited by the cytokines and supported by either intact
fibronectin or the 110-kD polypeptide (Fig 7C). In contrast, dual
blockade with MoAbs 333 and IST-6 completely impeded Sc-1 cell
chemotaxis (Fig 7C). Finally, an additional discrepancy observed
between the inhibitory effects exerted by the anti-central cell-binding
domain antibodies on cell adhesion and cytokine-induced motility was
the ability of the anti-3Fn9 antibody HFN-7 to abrogate chemotaxis when
used in combination with MoAb 333 (Fig 7C). This finding indicated the
participation of a 3Fn9 binding site in the regulation of 4
integrin-mediated leukocyte movement on fibronectin.
| |
DISCUSSION |
The present study shows that certain highly malignant,
41high/47high/51low
human B-lymphoma cells can recognize the central cell-binding region of
fibronectin constitutively, ie, without any prior artificial cellular
activation or immunological manipulation of their integrin receptor
complexes. In fact, Sc-1 and Ci-1 cells, but not a number of other
neoplastic leukocytes, were found to use this region of the molecule to
functionally complement the alternatively spliced carboxyl-terminal
IIICS region, as summarized in Fig 8. In
the case of initial cell tethering, a tentative assessment of this complementarity indicated that the relative contribution of these two
cell-binding domains was 70% to 30% in favor of the IIICS (when based
on a quantification of the binding activity in molar equivalents). The
corresponding relative contribution of the 41 and 47
integrins in this adhesion phenomenon was estimated to be 80% to 20%,
as suggested by the results obtained after differential antibody
blockade of the two integrins. Although acting in concert, the
receptors' capability to mediate cell interaction with the two domains
seemed to differ in that the 41 integrin preferentially mediated
binding to the CS-1 site, whereas the 47 was particularly important for the recognition of the central cell-binding domain. Both
integrin receptors mediated binding to at least two sites within this
latter domain: the RGD site and a synergistic site distinct from the
previously reported PHSRN and likely to reside within the 3Fn8 module
or at boundary region between 3Fn8 and 3Fn9. Even in this case, there
was a differential preference for binding sites in that the 41
preferentially reacted with the RGD sequence and the 47 proved to
be the pivotal receptor in the recognition of the putative 3Fn8
synergistic site (Fig 8).
View larger version (56K):
[in this window]
[in a new window]
| Fig 8.
Suggested model for the relative contributions of the
cooperating 41 and 47 integrins and their cognate binding
sites in the process of cell adhesion and migration on fibronectin.
Both receptors interchangeably recognize at least 4 different binding
sites residing within the central cell-binding and IIICS alternative
spliced domains and acting in concert. Within the former, three 3Fn
modules are the ones primarily implicated, ie, 3Fn8-10. Each of these
modules embodies at least one binding site, which is differentially
recognized by the integrins during the process of leukocyte adhesion
and chemotactic migration. The RGD site is the active one of 3Fn10,
whereas the synergistic sites residing within 3Fn8/3Fn9 remain
unidentified. The CS-1 site seems to be the only active one of the
IIICS domain. Both the relative contributions of the two domains and of
the integrin receptors recognizing the corresponding sites within these
domains differ in the processes of adhesion and migration. In the case
of cell adhesion, 47 shows a bias towards the putative novel
synergistic site within 3Fn8, whereas 41 preferentially binds to
the RGD site of 3Fn10.
|
|
In contrast to initial cell adhesion, the relative contribution of the
two fibronectin cell-binding domains in IL-1/IL-2-induced chemotaxis was virtually equal, and none of the cell- binding domains
alone was capable of supporting levels of cell movement paralleling
that sustained by intact fibronectin. In the chemotactic motility
process, the integrins seemed equally important in mediating the
migratory response, exhibiting a fully interchangeable binding to the
IIICS and central cell-binding domains. However, the mode by which the
cytokine-induced motility was supported by the central cell-binding
domain of fibronectin differed from the initial cell adhesion event. In
fact, the former involved multiple synergizing binding sites within the
3Fn8-10 modules (Fig 8).
The ability of B lymphomas, notably certain Burkitt's type lymphomas
such as Ramos and Daudi cells, to recognize the central cell-binding
domain of fibronectin through their 4 integrins has previously been
documented.15,16 However, this activity was demonstrated to
require the currently debated, experimental antibody-mediated
hyperactivation of 1 integrin receptors.29 It is now
evident that a rather nonspecific mechanism of 3Fn module recognition
can be elicited in cultured cells by antibody activation or by
Mn2+ binding to the integrin subunits, irrespectively of
the site or matrix molecule in which these modules are presented to the cells.30 Thus, we have identified here a novel
physiological mechanism of 4-integrin interaction with the central
cell-binding domain of fibronectin that may operate in untreated
neoplastic B leukocytes. Moreover, our data establish that recognition
of the RGD-containing region of fibronectin by constitutively active 4 integrins cannot be perturbed by soluble RGD-containing peptides, suggesting that, in contrast to 51 and v integrins, 4
integrins require structural integrity of the RGD loop and its
surrounding sequences. The discrepancies observed between constitutive
versus artificially activated 4 integrins may also be due to
differences in the ligand-induced, conformation-dependent modulation of
the activity of these receptors.30 Alternatively, the
divergent binding properties may be related to cell phenotype-specific
regulation of these integrins, which confer on the receptors disparate
intrinsic capacities to bind with high avidity to the various sites in
the central cell-binding domain of fibronectin.
Our data suggest that 4 integrins display higher affinity for a
sequence(s) contained within the CS-1 peptide (when provided in soluble
form) than for the RGD sequence, when presented comparably in solution
phase. This finding is in accord with a recent study showing that
interconversion of the RGD cell-binding motif to LDV (contained by the
CS-1 peptide) retains a potent antagonistic effect on 41
integrins.31 Moreover, binding of constitutively active
41 integrins to CS-1 and VCAM-1 can be perturbed by a cyclic
peptide containing the RGD sequence.32,33
These binding interactions can further be inhibited up to 1,000-fold
more effectively by LDV-containing peptides synthesized in an analogous
cyclic configuration.31-33 Thus, taken together, these
previous observations and our results suggest that the actual
activation state of 4 integrins may determine their relative
affinity for synthetic peptides with different amino acid compositions
and/or tertiary structures in solution, and they imply the
following order of affinity: cyclic LDV > linear CS-1 > cyclic RGD > linear RGD peptides. Finally, because of the apparent high affinity
of the 4 integrin subunit for the LDV sequence, it may not be
surprising that the experimentally activated receptors can bind
efficiently to KLDAPT peptides,19 in which the valine to
alanine amino acid substitution may cause little or no change in the
structural configuration of the binding site.
In analogy with the well-studied 51-mediated leukocyte
interaction with fibronectin,8,10 we report that 4
integrin-dependent B-lymphoma binding to the central cell-binding
domain of fibronectin seems to involve at least two independent binding
sites. The latter conclusion derives from the observation that both
MoAb 333, reacting with an epitope localized within the 3Fn10/3Fn11
modules,3 and the 3Fn8-directed MoAb IST-6 partially
blocked cell adhesion to the central cell-binding domain when used
alone, but completely abrogated the cell-fibronectin interaction when
used in combination. The inhibitory effect of MoAb IST-6 identifies an
ancillary synergistic cell adhesion site within the 3Fn8 module or
possibly within the amino-terminal portion of the 3Fn9 module in the
region adjacent to the 3Fn8. Experiments involving the use of
antibodies against the 4 and 1 integrin subunits, and against the
47 heterodimer, showed that both receptor complexes were
cooperatively responsible for the B-cell lymphoma-fibronectin
interaction. However, their relative participation in this interaction
differed. In fact, the contribution of 47 to the initial adhesion
of cells to the central cell-binding domain was proportionally greater
than that to the carboxyl-terminal IIICS region, whereas the
corresponding relative contribution of the 41 integrin to the
binding to the two separate domains was equivalent. This relationship
was demonstrated by the twofold to fourfold difference in the f |
Top
Abstract
Introduction