From the
The laminin-derived synthetic peptide containing the SIKVAV
(Ser-Ile-Lys-Val-Ala-Val) amino acid sequence has been previously shown
to regulate tumor invasion, metastasis, and angiogenesis. Here, we
demonstate that this peptide also modulates human monocyte responses.
Moreover, the monocytic responses elicited by this peptide are
influenced by the culture conditions. When elutriated monocytes were
cultured on SIKVAV substrate or in suspension with this peptide, the
synthesis of prostaglandin E
Monocytes are recruited to sites of tissue injury or chronic
inflammation by cell-derived chemotactic factors and/or degraded
extracellular matrix components. Once at the site, the monocyte may
orchestrate the extent of connective tissue destruction, in part, by
the production and activation of a family of metalloproteinases that
degrade all the major components of the extracellular matrix (for
review, see Birkedal-Hansen et al. (1993)). In addition to
these matrix metalloproteinases (MMPs),
Matrix components have also been shown
to influence monocyte functions. Exposure of monocytes to type I
collagen, gelatin, and endothelial cell-derived basement membrane
induces the production of PGE
The glycoprotein laminin-1, a major
component of the basement membrane, is composed of three chains
(
We demonstrate in this report that the laminin peptide SIKVAV
increases the production of PGE
Monocytes were cultured under different conditions to
simulate situations that occur either in the blood stream or at sites
of tissue damage. When the monocytes were cultured in suspension,
SIKVAV stimulated the production of PGE
Cellular invasion studies using the
U937 monoblastic cell line suggest that gelatinase B may be the
protease utilized by monocytes to transverse the extracellular matrix
and enter inflammatory sites (Watanabe et al., 1993). Once at
the inflammatory site, the production of interstitial collagenase by
these cells is pivotal for initiating the degradation of fibrillar
collagen in the damaged matrix. Stimulation of monocyte interstitial
collagenase, gelatinase B, PGE
The ability of degraded extracellular matrix
molecules to elicit an inflammatory response by human monocytes, such
as the production of tumor necrosis factor, has been observed with
fibronectin and its fragments (Beezhold and Personius, 1992; Chang
et al., 1993). Other studies have reported that the manner in
which the monocytes are cultured may modulate their responses to
extracellular matrix molecules (Kohn and Klingemann, 1991). Native and
denatured collagens have also been reported to induce the production of
arachidonic acid metabolites by monocytes (Gudewicz et al.,
1994) and interstitial collagenase by alveolar macrophages (Shapiro
et al., 1993). Collagen and collagen fragments also enhance
chemotaxis, respiratory burst, gelatinase, and elastase production by
alveolar macrophages (Laskin et al., 1994). Although native
laminin has been previously observed to be inert in stimulating
monocyte function (Shapiro et al., 1993; Gudewicz et
al., 1994), we show here that the laminin-derived amino acid
sequence SIKVAV, by itself, induces an inflammatory response from human
monocytes. This suggests that this site of laminin contributes not only
to promote angiogenesis but also to potentiate monocytic inflammatory
responses.
Previously, SIKVAV has been reported to promote
angiogenesis by inducing endothelial cell adhesion, migration, and
invasion (Kibbey et al., 1992). In a murine model, injection
of matrigel along with the SIKVAV peptide results in vascularization of
the matrigel plug. This effect may be partly due to the infiltration of
polymorphonuclear cells (PMNs) and subsequently monocytes (Kibbey
et al., 1994) into the SIKVAV-containing matrigel plug. If
these mice are made neutropenic, the angiogenesis in the matrigel plug
containing SIKVAV is greatly reduced. Moreover, we also demonstrated
that PMNs have the capacity to release proteases that degrade intact
laminin molecules. It is possible that the PMNs, one of the first cells
to arrive at the inflammatory site, initiate the degradation of matrix
components such as collagen and laminin. The degradation of laminin and
subsequent exposure of peptides containing the SIKVAV sequence may
potentiate the inflammatory response by infiltrating monocytes. The
ability of laminin peptides to modulate the production of
PGE
We thank Dr. William Stetler-Stevenson of the
Extracellular Matrix Pathology, Laboratory of Pathology, National
Cancer Institute for providing the antibodies against interstitial
collagenase and gelatinase B and Susan Hopkinson for her excellent
technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, interstitial collagenase, and
gelatinase B was induced and was further enhanced in the presence of
concanavalin A (ConA). However, when monocytes were adhered before
adding soluble SIKVAV, the peptide alone failed to induce the
production of prostaglandin E
or matrix metalloproteinases.
If adherent monocytes were exposed to SIKVAV in the presence of ConA,
this peptide enhanced the ConA induced production of these mediators.
In contrast to SIKVAV, the intact laminin molecule failed to influence
these monocyte responses. This is the first demonstration that a
laminin derived peptide is capable of inducing or enhancing monocyte
inflammatory responses that may influence a number of biological
activities such as wound healing or excessive connective tissue
destruction associated with chronic inflammation.
(
)
monocytes secrete inflammatory mediators such as
prostaglandin E
(PGE
), tumor necrosis factor,
and interleukin-1 (IL-1) which influence the production of MMPs by
other cells. Treatment with lipopolysaccharide or concanavalin A (ConA)
induces monocytes to produce PGE
which leads to the
production of MMPs (Wahl and Lampel, 1987). Conversely, agents that
suppress monocyte production of PGE
, such as
anti-inflammatory drugs (Wahl and Lampel, 1987), interferon-
(Wahl
et al., 1990), IL-4 (Corcoran et al., 1992), and
IL-10 (Mertz et al., 1994), also inhibit the production of
MMPs. Thus, the synthesis of MMPs by monocytes is dependent on
PGE
production.
(Gudewicz et al.,
1994). Native and denatured type I collagen also induce the production
of monocyte interstitial collagenase (Shapiro et al., 1993) as
well as a number of responses by alveolar macrophages including
chemotaxis, oxidative burst, cytotoxicity, and production of elastase
and gelatinase (Laskin et al., 1994). Although intact laminin
does not induce the production of MMPs by macrophages/monocytes
(Shapiro et al., 1993), we examined whether specific sites on
laminin, possibly exposed during degradation of this molecule, could
modulate monocyte function.
1,
1, and
1) which form a covalent cross-like molecule
(Burgson et al., 1994). Laminin promotes cell adhesion,
migration, differentiation, and tumor metastasis. Several sites on the
laminin molecule that mediate these biological responses have been
identified at the synthetic peptide level (Kleinman et al.,
1993). One of these, the SIKVAV site (Ser-Ile-Lys-Val-Ala-Val), located
on the long arm of the laminin
1 chain has been shown to promote
attachment, migration, angiogenesis (Kibbey et al., 1994),
protease production, tumor growth, and metastasis (Kanemoto et
al., 1991; Tashiro et al., 1991; Grant et al.,
1992; Kibbey et al., 1992; Sweeney et al., 1991;
Stack et al., 1991). Thus, this peptide has the potential to
regulate a number of cellular responses in a cell type-specific manner.
, interstitial collagenase
(EC 3.4.24.7) and gelatinase B (EC 3.4.24.35) by monocytes. These data
suggest that laminin fragments may play an important role in the
resolution of tissue damage at a wound site and/or the exacerbation of
connective tissue destruction associated with chronic inflammatory
lesions.
Monocyte Purification
Peripheral blood
mononuclear cells were obtained by leukapheresis of normal volunteers
at the Department of Transfusion Medicine at the National Institutes of
Health. The monocytes in the mononuclear fraction were enriched to
90-95% by counterflow centrifugal elutriation using
endotoxin-free reagents as described previously (Wahl et al.,
1984).
Laminin Peptide Production and Purification
The
1 chain-derived SIKVAV-containing peptide (amino acids
2099-2105; CRKQAASIKVAVS) was synthesized, purified as described
previously (Graf et al., 1987) and endotoxin-filtered. Control
peptides include the SHA-7 peptide (CSRARKQAASGKVAVSADR) which contains
a single amino acid substitution in the SIKVAV active site, and YOSH-3
(CRTDEGEKKCGCPGC) which is an inactive sequence from the
1 chain
of laminin. These peptides tested negative for endotoxin by the limulus
amebocyte lysate assay.
Culture Conditions
Monocytes (20
10
/4 ml) were cultured in Dulbecco's modified
Eagle's medium (BioWhittaker) for 48 h at 37 °C under the
following conditions: in suspension in 15-ml polypropylene tubes
(Falcon) along with the peptides, in 60-mm tissue culture dishes that
had been previously coated with peptides, or adhered to 60-mm culture
dishes prior to the addition of peptides. Peptides were coated onto
60-mm tissue culture dishes by drying sterile phosphate-buffered saline
containing the laminin peptides. Phosphate-buffered saline that did not
contain peptide was used as the control.
Monocyte PGE
After 48 h of incubation under the culture
conditions indicated above, the conditioned media were harvested and
the levels of PGE and Matrix Metalloproteinase
Assays
in the supernatants were determined by
radioimmunoassay (Wahl, 1981). Western blot analysis for the MMPs was
performed as described previously (Corcoran et al., 1992).
Briefly, the secreted proteins in the remaining supernatants were
ethanol-precipitated, separated on reducing 8-16% Tris/glycine
polyacrylamide gels and transferred onto a nitrocellulose membrane. A
1:100 dilution of antibody 110, which recognizes the latent and active
forms of gelatinase B (92-kDa type IV collagenase), and antibody 125,
which recognizes the active forms of interstitial collagenase was used
followed by an incubation with 1:16,000 dilution of protein
G-horseradish peroxidase. The MMP proteins were visualized by
chemoluminescence (ECL kit, Amersham Corp.) and the intensity of the
signal quantitated using Image 1.45 software and an Arcus scanner.
Effect of Substrate-bound SIKVAV on Monocyte/Macrophage
Function
Freshly isolated human monocytes were added to tissue
culture dishes that were previously coated with the laminin peptides.
After 48 h, the conditioned media were evaluated for the presence of
inflammatory mediators including interstitial collagenase, gelatinase
B, and PGE. As determined by Western blot analysis,
monocytes cultured on either 100 µg or 200 µg/well of
SIKVAV-containing peptide induced the production of interstitial
collagenase which was predominantly the 47- and 42-kDa catalytically
active species (Fig. 1). Induction of interstitial collagenase
was not observed when monocytes were cultured on YOSH-3, an inactive
peptide sequence from the
1 chain of laminin, or on uncoated
plastic. Native laminin did not induce the production of these
mediators (data not shown). As expected, 20 µg/ml of ConA
stimulated production of interstitial collagenase on nontreated dishes.
Adding 20 µg/ml of ConA to the monocytes cultured on SIKVAV-coated
dishes further enhanced the production of interstitial collagenase over
that produced by monocytes cultured in the presence of SIKVAV alone
(Fig. 1).
Figure 1:
Effect of
substrate bound laminin SIKVAV peptide on monocyte interstitial
collagenase production. Elutriated monocytes (20
10
/4 ml) were added to 60 mm tissue culture wells that were
previously coated with no peptide or 100-200 µg of either
SIKVAV or YOSH-3 per well. The monocytes were incubated in the presence
or absence of 20 µg/ml of ConA for 48 h. The proteins in the
conditioned media were ethanol precipitated and analyzed by Western
blot with an antibody against interstitial collagenase as described in
the Materials and Methods. This Western shows both the 42 and 47-kDa
catalytically active collagenase protein.
Monocytes cultured on SIKVAV-coated dishes also
produced gelatinase B (Fig. 2). A control peptide, SHA-7, which
contains a single amino acid substitution (SGKVAV) in the biologically
active site, did not induce the production of this enzyme. These
results were also confirmed by gelatin zymography (data not shown).
Since PGE production correlates with the production of
monocyte MMPs (Corcoran et al., 1992), the levels of this
mediator were also determined. The production of PGE
(4.03
± 1.4 ng/ml) by monocytes cultured on substrate-bound SIKVAV
peptide (100 µg/ml) was 4-fold higher compared with no peptide
treatment (1 ± 0.4 ng/ml). Addition of 1 µg/ml of ConA to
the SIKVAV-coated dishes induced more than a 2-fold increase in
PGE
production (22.5 ± 2.4 ng/ml) over that observed
from cells cultured on plastic and stimulated with ConA alone (9.9
± 1.7 ng/ml).
Figure 2:
Effect of substrate bound laminin SIKVAV
peptide on monocyte production of latent gelatinase B. Human monocytes
(20 10
/4 ml) were added to 60 mm wells that had
been previously treated with no peptide or 200-400 µg of
SIKVAV peptide per well. The proteins secreted after a 48 h incubation
were ethanol precipitated and Western blotted with an antibody against
92-kDa gelatinase B as described in the Materials and
Methods.
Effect of Soluble SIKVAV on Adhered
Monocytes
Monocytes cultured on tissue culture plates prior to
addition of soluble SIKVAV peptide did not produce interstitial
collagenase (Fig. 3). However, if the adhered monocytes were
treated with 1 µg/ml ConA as well as soluble SIKVAV (400
µg/ml), the production of interstitial collagenase was
significantly enhanced over that observed with ConA (1 µg/ml)
treatment alone (Fig. 3). Similarly, the addition of SIKVAV
peptide alone to adherent monocytes did not induce PGE production. However, the PGE
produced in response to
1 µg/ml of ConA (6.5 ± 1.9 ng/ml versus control,
0.85 ± 0.2 ng/ml) was enhanced 4-5-fold (29 ± 7
ng/ml) by 100 µg/ml soluble SIKVAV peptide.
Figure 3:
Modulation of interstitial collagenase
production by adherent monocytes with soluble laminin SIKVAV peptide.
Freshly isolated monocytes (20 10
/4 ml) were
adhered to 60 mm tissue culture dishes for 30 min prior to addition of
soluble peptides. After a 48 h incubation period, the proteins in the
supernatants were ethanol precipitated and Western blotted with an
antibody against interstitial collagenase as described in Materials and
Methods.
Effect of Soluble SIKVAV Added to Monocytes in
Suspension
When the monocytes were cultured with the peptides in
suspension, SIKVAV alone was also shown to induce interstitial
collagenase in a dose-dependent manner (Fig. 4). While soluble
SIKVAV alone (200 or 400 µg/ml) induced the production of
interstitial collagenase, when a suboptimal stimulatory dose of ConA (1
µg/ml) was added with SIKVAV there was a synergistic induction of
interstitial collagenase (Fig. 4). There was a positive
correlation between MMP and PGE production. Suspended
monocytes treated with soluble SIKVAV (100 µg/ml) peptide had a
5-fold increase in PGE
levels (8.5 ± 0.1 ng/ml) as
compared with untreated cells (1.6 ± 0.9 ng/ml).
Figure 4:
Effect of soluble laminin SIKVAV peptide
on interstitial collagenase production by monocytes cultured in
suspension. SIKVAV laminin peptide was added to monocytes (20
10
/4 ml) in suspension and cultured for 48 h in the absence
or presence of ConA (1 µg/ml). Stimulation with 20 µg/ml of
ConA is also shown as a positive control. The conditioned media were
ethanol precipitated and Western blotted with an antibody against
interstitial collagenase as described in the Materials and
Methods.
Soluble
SIKVAV (100 µg/ml) peptide also elicited the production of latent
gelatinase B by monocytes cultured in suspension (Fig. 5). In the
presence of ConA (20 µg/ml), SIKVAV enhanced by 4-5-fold the
production of the 92-kDa gelatinase B over that induced with ConA
treatment alone (Fig. 5). Additionally, the 84-kDa
proteolytically active product of gelatinase B was detected when
monocytes were treated with ConA and SIKVAV.
Figure 5:
Effect of soluble laminin SIKVAV peptide
on the production of latent and active gelatinase B by monocytes
cultured in suspension. Human monocytes (20 10
/4
ml) were cultured in suspension for 48 h in the presence or absence of
SIKVAV peptide and ConA (20 µg/ml) as indicated. The proteins in
the media were ethanol precipitated and Western blotted using an
antibody against gelatinase B as described in the Materials and
Methods. This Western shows both the latent 92-kDa species as well as
the catalytically active 84-kDa product.
, interstitial
collagenase, and gelatinase B in the absence of a primary stimulus
(ConA). In the presence of suboptimal concentrations of ConA, monocytes
cultured in suspension responded to SIKVAV by inducing a greater
enhancement of monocyte PGE
and MMPs. Substrate-bound
SIKVAV also directly elicited the production of PGE
,
interstitial collagenase and gelatinase B, and enhanced these responses
in the presence of a suboptimal concentration of ConA. However, when
monocytes were adhered, SIKVAV failed to induce MMP and PGE
production, but enhanced these responses when they were initiated
by ConA. The fact that SIKVAV can directly induce monocyte protease
production agrees with previous studies on endothelial cells (Grant
et al., 1992). The differential responses to SIKVAV observed
under different culture conditions may be important in understanding
the control mechanisms that limit monocyte activation at sites of
inflammation and tissue damage.
, and possibly other monocyte
functions by SIKVAV may be important in promoting tissue repair during
wound healing.
, interstitial collagenase, as well as the gelatinase B
by monocytes could play an important role in their ability to
transverse basement membranes. Thus, laminin fragments containing the
SIKVAV sequence may contribute to the inflammatory response at sites of
wound healing. Additionally, laminin fragments containing the SIKVAV
sequence may contribute to the excessive induction of
metalloproteinases that ultimately result in destruction of connective
tissue associated with chronic inflammatory lesions.
, prostaglandin
E
; ConA, concanavalin A; IL-1, interleukin-1; PMNs,
polymorphonuclear cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.