(Received for publication, August 5, 1996, and in revised form, January 8, 1997)
From the Department of Pathology, the
§ Department of Cell Biology & Anatomy and the Center of
Vascular Biology, Cornell University Medical College,
New York, New York 10021
Urokinase-type plasminogen activator (uPA) and
92-kDa matrix metalloproteinase (MMP-9) expression by RAW264.7
macrophages were up-regulated when plated on extracellular matrices.
Collagen IV, fibronectin, and tenascin stimulated macrophages' MMP-9
expression. In contrast, laminin stimulated both uPA and MMP-9
expression in a dose- and time-dependent manner. The
increase in macrophage uPA activity was preceded by an increase in
their steady state levels of uPA mRNA. Laminin-induced uPA
expression was most pronounced in RAW264.7 macrophages followed by
THP-1 monocytes, J774A.1 macrophages, and bone marrow-derived
macrophages. Neither laminin nor matrix induced alterations in THP-1
monocyte expression of plasminogen activator inhibitor, tissue
inhibitor of metalloproteinase (TIMP)-1 or TIMP-2. Synthetic laminin
peptides were utilized to identify the laminin domain(s) responsible
for induction of uPA expression. Peptides derived from the 1 chain
of laminin had no effect on macrophage uPA expression, whereas SIKVAV,
derived from
1 chain, stimulated uPA expression 20-fold.
Preincubation of THP-1 monocytes with a monoclonal antibody directed
against the
6 subunit of the
6
1 laminin receptor blocked
matrix induction of uPA without affecting the induction of MMP-9. These
results demonstrate that macrophage binding to laminin plays an
important role in the regulation of their degradative phenotype via the
up-regulation of uPA and MMP-9.
Macrophages utilize serine proteinases, metalloproteinases, and cysteine proteinases to degrade extracellular matrix (1-4). Principal among these proteinases is the serine proteinase uPA1 (5). uPA cleaves the Arg560-Val561 bond of plasminogen, thereby generating plasmin, a serine protease with wide substrate specificities (5). Plasmin binds to and degrades several components of the extracellular matrix including fibronectin, laminin, and proteoglycan core protein (6, 7). Moreover, plasmin activates a family of MMPs (8).
In addition to the cooperative role proteinases have in matrix
degradation, they participate in the regulation of several unexpected
biological processes. uPA and plasmin activate hepatocyte growth factor
(9) and latent transforming growth factor (10), respectively. In
addition, plasmin liberates cell- and matrix-bound basic fibroblast
growth factor and transforming growth factor
(11-13). MMPs
activate pro-interleukin-1 (14) and release from the cell surface tumor
necrosis factor, tumor necrosis factor receptor, colony stimulating
factor-1, Fas ligand, and interleukin-6 receptor (15-17).
Consequently, cellular regulation of the proteinase cascade has effects
beyond matrix degradation.
Macrophages localize and regulate plasminogen activation via their expression of uPA, PAI, uPA receptor, and several binding sites/receptors for plasmin(ogen) (18). The expression of uPA, uPA receptor, and PAI is regulated by a variety of cytokines and growth factors (19-22). Likewise, macrophage MMP expression is highly regulated. Macrophages express interstitial collagenase (MMP-1), 92-kDa gelatinase (MMP-9), 72-kDa gelatinase (MMP-2), and stromelysin (MMP-3) (2, 23). The expression of MMPs and their inhibitor, TIMP, is also regulated by cytokines (24-27). In addition, structural components of the matrix regulate macrophage MMP expression. For example, native and denatured collagen types I and III stimulated MMP-1 expression by alveolar macrophages (28), whereas other matrix components including laminin, fibronectin, and elastin were ineffective (28). In other studies, it was demonstrated that fibronectin, laminin, and entactin stimulate production of 96- and 58-kDa MMPs by murine peritoneal macrophages (29). Finally, the laminin peptide SIKVAV induced human monocyte/macrophage MMP-1 and MMP-9 expression (30). Taken together, these data demonstrate that macrophage-matrix interactions are an important factor in their expression of a degradative phenotype and tissue remodeling.
In experiments reported here, we have determined whether macrophage uPA
expression, a pivotal component of the proteinase cascade, is regulated
by the extracellular matrix. Results demonstrate that macrophage uPA
and MMP-9 expression are up-regulated when cultured on extracellular
matrix. The matrix component responsible for the increase in uPA and
MMP-9 expression is laminin. Collagen, fibronectin, and tenascin
up-regulated MMP-9 expression without affecting uPA expression. The
effect of laminin on macrophage uPA expression was mediated by a
portion of the long arm of laminin containing the sequence SIKVAV and
was blocked by antibody to the 6
1 (VLA-6) integrin.
Murine RAW264.7 and J774A.1 macrophages and human THP-1 monocytes were obtained from American Type Tissue Culture (Rockville, MD). RAW264.7 and J774A.1 cells were maintained as adherent cultures in Roswell Park Memorial Medium (RPMI; without HEPES) supplemented with 10% Cellect Gold fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 µg/ml), and 4 mM glutamine (Life Technologies, Inc.). THP-1 monocytes were maintained in suspension in the same medium. Human bone marrow-derived macrophages were obtained from Dr. Shahin Rafii, Cornell Medical College. When assessed by immunoperoxidase staining, bone marrow-derived macrophages were highly reactive with monoclonal antibodies against the human macrophage markers CD68 (DAKO, Carpinteria, CA) (31) and HAM56 (DAKO) (32).
Preparation of Matrix-coated PlatesTissue culture plates (24 well) were coated with Matrigel (Collaborative Biomedical Products, Bedford, MA), a basement membrane matrix derived from Englbreth-Holm-Swarm mouse sarcoma (33). Matrigel was thawed at 4 °C, diluted to 1 mg/ml with macrophage serum-free medium (MSFM) (Life Technologies, Inc.), and aliquoted into wells (0.5 ml/well). Plates were incubated at room temperature for 1 h. In other experiments, tissue culture plates were incubated overnight at 4 °C with 10 µg/ml laminin (Life Technologies, Inc.), fibronectin (Life Technologies, Inc.), collagen IV (Calbiochem), or tenascin (Life Technologies, Inc.). The next day, wells were washed with DPBS prior to the addition of cells.
Effect of Matrix and Matrix Components on uPA and MMP ExpressionRAW264.7, J774A.1, and bone marrow-derived macrophages were mechanically harvested and collected by centrifugation (400 × g, 10 min). Cells were washed with DPBS (3 ×) and suspended in MSFM supplemented with antibiotics. Cells were aliquoted into wells previously treated with Matrigel or purified matrix components as described above. At the indicated time, media were removed and assayed for uPA and MMP activities.
Effect of Synthetic Laminin Peptides on Macrophage uPA and MMP ExpressionLaminin peptides CDPGYIGRS and YIGSR were obtained
from Sigma. SIKVAV was purchased from Bio-Synthesis
(Lewisville, TX). CDPGYIGR and YIGSR were dissolved in water (1 mg/ml).
SIKVAV was suspended in DPBS (5 mg/ml), sonicated, and vortexed until
dissolved. Peptides were sterile-filtered and stored at 20 °C.
Cells were incubated with MSFM containing 10-50 µg/ml synthetic
laminin peptides. The next day media were recovered and assayed for uPA
and MMP activity.
Monoclonal anti-6 (CDw49f) IgG was
obtained from BioDesign (Kennebunk, ME). Monoclonal anti-Fc
RIII
(CD16) IgG was obtained from Immunotech (Westbrook, ME). Antibodies
were dialyzed against DPBS to remove azide and stored at
20 °C.
Human THP-1 monocytes were harvested by centrifugation (400 × g, 10 min), washed in DPBS, and resuspended in MSFM
containing anti-
6 IgG or anti-Fc
RIII IgG. Cells were incubated 30 min in a microcentrifuge tube at 37 °C and then transferred to
matrix-coated wells. The next day, media were recovered and assayed for
uPA and MMP activity.
Plasminogen activator activity was quantitated utilizing a previously described modification of a sensitive functional assay for plasmin (34). Aliquots of conditioned media were added to microtiter wells containing 82 µl of DPBS, 0.05% Tween 20 containing 13 µg of the plasmin substrate D-Val-Leu-Lys-aminomethylcoumarin (Enzyme Systems Products, Dublin, CA) and 0.5 µg of bovine plasminogen (American Diagnostica, Greenwich, CT). Samples were mixed and incubated at 37 °C for 2.5 h. Cleavage of the substrate was monitored by measuring the increase in fluorescence in a Fluoroscan microplate reader (excitation, 330-380 nm; emission, 430-530 nm). Concentrations of uPA in the test samples were extrapolated from a standard curve utilizing high molecular weight uPA (American Diagnostica). Plasminogen activator activity in macrophage-conditioned media was completely inhibited when preincubated with a polyclonal anti-human uPA IgG (American Diagnostica) as described previously (35).
Northern Blot for uPA, PAI2, and TIMP-2 mRNA LevelsRNA was isolated from murine RAW264.7 macrophage and human THP-1 monocytes as described previously (35). RNA samples were electrophoresed in agarose, transferred to nylon membrane (Schleicher & Schuell), and hybridized with either a 32P-labeled murine cDNA for uPA (36; provided by Dr. J. Degen, Children's Hospital Research, Cincinati, OH), human cDNA for TIMP-2 (37; ATTC), or human cDNA for PAI2 (38; ATTC). Equal amounts of total RNA were loaded per lane as judged by UV inspection of ethidium bromide-stained agarose gels.
Zymographic Demonstration of Metalloproteinase ActivityConditioned media were concentrated in an ice bath utilizing an Amicon ultrafiltration chamber with a YM-10 (10-kDa cutoff membrane). SDS sample buffer without mercaptoethanol was added to the media samples and heated for 30 min at 37 °C. Samples and molecular weight markers were electrophoresed in a 10% polyacrylamide gel containing 0.1% gelatin. The gel was then washed (2 ×) in 2.5% Triton X-100 to remove SDS. The gel was incubated at 37 °C for 48 h in 200 mM NaCl containing 40 mM Tris-HCl and 10 mM CaCl2, pH 7.5, and stained with Coomassie Blue. The presence of gelatinolytic activity was identified as clear bands on a uniform blue background following destaining.
Western Blot for TIMP-1 AntigenTHP-1 monocyte conditioned
media were mixed with sample buffer containing -mercaptoethanol and
immersed in boiling water for 5 min. Recombinant TIMP-1 (Calbiochem)
and samples were electrophoresed in 4-15% polyacrylamide gradient
gels. Proteins were transferred to nitrocellulose membrane, following
which the membrane was blocked in TTBS containing 1% bovine serum
albumin for 1 h. Following two washes (TTBS), the membrane was
incubated with blocking buffer containing 1 µg/ml monoclonal
anti-TIMP-1 IgG (Calbiochem) overnight at 4 °C. The membrane was
washed (2 ×, TTBS) and incubated for 1 h with 0.5 µg/ml
biotinylated rabbit anti-mouse IgG (Pierce) in blocking buffer at room
temperature. The membrane was then washed (2 ×, TTBS) and incubated
1 h with avidin-biotin-horseradish peroxidase complexes (Pierce)
in DPBS, 0.5% Tween-20. Bound horseradish peroxidase was detected
utilizing enhanced chemiluminescence (Amersham Corp.) as per
manufacturer's instructions.
Cellular expression of plasminogen
activators and metalloproteinases cooperate in matrix degradation (8,
39). In these experiments, we determined the ability of matrix to
regulate macrophage expression of both uPA and metalloproteinases. For
these purposes, RAW264.7 macrophages were cultured overnight on either
plastic or extracellular matrix-coated (Matrigel) plastic. Conditioned media from cells grown on plastic contained 97 ± 8 milliunits of
uPA (Fig. 1). When cultured on matrix, uPA levels in
conditioned media increased >6-fold. To determine whether a soluble
factor present in the matrix was responsible for the observed increase in uPA activity, matrices were incubated overnight with media alone.
The matrix conditioned media were recovered and added to cells grown on
plastic. As observed in Fig. 1, uPA expression by cells cultured on
plastic was not affected when incubated with matrix-conditioned media.
Likewise, matrix induction of macrophage uPA expression was not
affected when cells were incubated with matrix-conditioned media. These
data demonstrate that an insoluble portion of the matrix was
responsible for the observed matrix induction of RAW264.7 macrophage
uPA expression.
Metalloproteinase activity in macrophage-conditioned media was
determined utilizing SDS-polyacrylamide gel electrophoresis zymography.
The metalloproteinases degrade gelatin incorporated into a
polyacrylamide gel. The degraded areas appear unstained following
staining with Coomassie Blue. Conditioned media from cells grown on
plastic contained metalloproteinase activity which based on molecular
weight appears to be the 92-kDa gelatinase or MMP-9 (Fig.
2). When cultured on matrix-coated plastic, MMP-9 levels
in macrophage-conditioned media increased severalfold.
Laminin Up-regulates Macrophage uPA and MMP-9 Expression
A
series of experiments were initiated to identify the component(s) of
the matrix that were responsible for the observed increase in RAW264.7
macrophage uPA and MMP-9 activities. As seen in Fig. 3,
insoluble or soluble collagen IV, fibronectin, and tenascin had little
or no effect on uPA expression. In contrast, uPA expresson by cells
cultured on laminin-coated plastic was increased 5-fold over controls.
When laminin was added to cells in the fluid phase, uPA expression was
increased >100-fold.
We next compared the effect of laminin on uPA expression by other murine and human monocyte/macrophage cells. Levels of constitutively expressed uPA in conditioned media derived from the control cells varied widely (3-68 milliunits) (Table I). Following an overnight incubation with laminin, uPA levels in conditioned media from all macrophages examined were increased. The increase in uPA activity in conditioned media of macrophages incubated with laminin was most pronounced in murine RAW264.7 macrophages (313-fold) followed by human THP-1 monocytes (41-fold), murine J774A.1 macrophages (18-fold), and human bone marrow-derived macrophages (6-fold).
|
Laminin's effect on RAW264.7 macrophage uPA expression was dose- and
time-dependent (Fig. 4). Significant
increases in uPA activity were observed in conditioned media when cells
are incubated with 1 µg/ml (1.1 nM) laminin. Maximal
stimulation was observed between 5 and 10 µg/ml. The effect of
laminin on macrophage uPA expression was rapid. A significant increase
in uPA activity was observed in conditioned media 2 h following
the addition of 5 µg/ml laminin (5.5 nM). Furthermore,
uPA activity in conditioned media continued to increase over the
experimental period (24 h). For comparison purposes, uPA activity in 24 h-conditioned media derived from control cells is presented.
The time-dependent increase in macrophage uPA expression
following exposure to laminin was preceded by an increase in the steady
state levels of uPA mRNA (Fig. 5). Steady state
levels of uPA mRNA were increased 1 h following exposure of
macrophages to laminin. uPA mRNA levels continued to rise over the
next hour and were 15-fold greater than controls at 24 h. In
contrast, laminin had no effect on steady state levels of
glyceraldehyde-3-phosphate dehydrogenase mRNA. The sustained effect
of laminin on uPA mRNA levels is in contrast to the transient
effect observed with phorbol myristate acetate and other agonists of
RAW264.7 uPA expression (35).
We next determined whether laminin up-regulated RAW264.7 macrophage
metalloproteinase expression. RAW264.7 cells were incubated overnight
with 0.1-10 µg/ml (0.11-11.1 nM) laminin. As seen in Fig. 6, control cell media expressed small amounts of
MMP-9 activity. Following incubation with laminin, the expression of
MMP-9 was increased in a dose-dependent manner.
Laminin-induced MMP-9 activity was visible at 4 h and increased
over the remainder of the experimental period (20 h)
(Fig. 6). In contrast to selective effect of laminin on
macrophage uPA expression, all matrix components examined stimulated MMP-9 expression by RAW264.7 cells (data not shown). Taken together, these data suggest that when cells were plated on basement
membrane-derived extracellular matrix, laminin up-regulates macrophage
uPA and MMP-9 expression, whereas several matrix components up-regulate the expression of MMP-9. These findings are significant since the
expression of both uPA and MMP-9 have been demonstrated to play a role
in cellular migration through matrix (40-43).
Macrophage Expression of TIMP-1, TIMP-2, and PAI2 Are Unaffected by Laminin
The effect of laminin on macrophage expression of the protease inhibitors TIMP-1, TIMP-2, and PAI2 was determined utilizing Northern and Western blots. Incubation of THP-1 monocytes with laminin for 24 h had no effect on the steady state levels of the 3.5- and 1.0-kilobase TIMP-2 mRNA transcripts (44; data not shown). Likewise, no change in TIMP-2 mRNA levels were observed when THP-1 monocytes were incubated on Matrigel for 2, 6, or 24 h. Levels of TIMP-1 protein (28 kDa) expressed by THP-1 monocytes incubated with either laminin or Matrigel for 24 h were unchanged as compared with cells incubated on plastic.
PAI2 is the predominant PAI isoform expressed by macrophages (20). Incubation of human THP-1 monocytes with laminin for 24 h had no effect on the steady state levels of the 2-kilobase PAI2 mRNA transcript (data not shown). Likewise, no change in PAI2 mRNA levels were observed when THP-1 monocytes were incubated on Matrigel for 2, 6, and 24 h. Therefore, it appears that purified laminin as well as intact matrix selectively induce macrophage uPA and MMP expression.
Laminin SIKVAV Peptide Up-regulates Macrophage uPA ExpressionWe have utilized synthetic laminin peptides to
identify the laminin domain(s) responsible for the induction of
macrophage uPA expression. CDPGYIGSR and YIGSR are derived from the
short arm of the 1 chain of laminin (45) and are reported to promote cell attachment (46, 47). SIKVAV is derived from the long arm of
laminin's
1 chain (45). It is reported to promote adhesion, cell
spreading, and stimulate tumor cell and monocyte MMP expression (30,
48). As seen in Table II, incubation of RAW264.7
macrophages with 10-50 µg/ml CDPGYIGSR and YIGSR had no effect on
uPA expression. In contrast to peptides derived from the short arm of
laminin, incubation of macrophages with SIKVAV stimulated their uPA
expression 20-fold. When RAW264.7 macrophages were incubated with a
scrambled peptide (IVKVSA) no increase in uPA expression was observed
(data not shown).
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The binding of cells to laminin is mediated by several
integrins and a nonintegrin receptor (49). E8 is an elastase-generated laminin fragment that contains a portion of the 1 chain containing the SIKVAV sequence (45). Cell binding to laminin fragment E8 is
mediated by
6
1 (VLA-6) integrin (50, 51). Since it was demonstrated that SIKVAV stimulates macrophage uPA expression (Table
II), we determined the effect of an antibody to the integrin
6
subunit on SIKVAV and matrix induction of uPA. THP-1 monocytes were
preincubated with monoclonal antibody (5 µg/ml) directed against the
integrin
6 subunit (CDw49f) before adding SIKVAV (50 µg/ml). Following an overnight incubation, media derived from control
cells contained 10 ± 0.3 milliunits uPA/2.5 × 105 cells (mean ± S.E.; n = 4). uPA
expression by cells incubated with SIKVAV peptide was increased
~5-fold (47.5 ± 0.6 milliunits). When cells were preincubated
with monoclonal anti-CDw49f IgG (2.5 µg/ml),
peptide-induced uPA expression was completely blocked (11 ± 0.5 milliunits), whereas preincubation of cells with monoclonal anti-CD16
IgG (Fc
RIII) had no effect. These data demonstrate that binding of
the SIKVAV peptide to the
6
1 integrin mediates laminin induction
of uPA expression.
However, the above data do not exclude the possibility that laminin
binding to other integrins may also regulate uPA expression. Therefore,
THP-1 monocytes were incubated with anti-CDw49f before adding to matrix-coated wells. As expected, uPA expression by THP-1
monocytes grown in matrix-coated wells was up-regulated severalfold
(Fig. 7). Preincubation with 2.5 µg/ml anti-CDw49f IgG
inhibited matrix induction of uPA expression 90% without affecting adhesion. At 5 µg/ml, anti-6 IgG completely blocked matrix-induced uPA expression. The control antibody, anti-CD16 IgG, did not
significantly reduce matrix-induced uPA expression at 2.5 µg/ml;
however, at 5 µg/ml there was a 25% reduction. Preincubation with
anti-CDw49f IgG or anti-CD16 IgG had no effect on matrix
induction of MMP-9 expression (data not shown). Taken together, these
data demonstrate that matrix induction of uPA is via laminin binding to
macrophage
6
1 integrin.
The signal transduction pathways by which laminin engagement of the
6
1 integrin induces macrophage uPA and MMP expression are not
known. However, induction of macrophage MMP by collagen and the SIKVAV
peptide is dependent on prostaglandin E2 synthesis and
blocked by the cyclooxygenase inhibitor indomethacin (24, 28, 30). In
preliminary studies, inhibitors of tyrosine kinase (herbimycin A) and
protein kinase C (calphostin C) blocked laminin-induced uPA expression.
In contrast, an inhibitor of cyclooxygenase (ibuprofen) had no effect
on laminin-induced uPA expression.
Macrophage tissue remodeling is regulated by the expression of matrix-degrading proteases and their inhibitors and cellular receptors to direct and localize proteolysis (1, 2, 52-55). The expression of these proteinases is regulated by cytokines and growth factors (22, 24, 25, 27). In addition, structural components of the extracellular matrix have been demonstrated to regulate MMP expression (28-30), bind plasminogen and PAI (56-58), and enhance the kinetics of plasminogen activation (59, 60). In studies reported here, we have demonstrated that macrophage expression of uPA and MMP-9 is up-regulated when cultured on basement membrane extracts. Of the purified matrix components examined, laminin up-regulated macrophage expression of both uPA and MMP-9, whereas collagen IV, fibronectin, and tenascin up-regulated MMP-9 without affecting uPA expression. To our knowledge, this is the first demonstration that macrophage uPA expression, a pivotal component of their tissue remodeling proteinase cascade, is up-regulated by laminin.
In these studies, macrophage uPA expression was quantitated utilizing a functional assay based on the ability of uPA to generate plasmin from exogenous plasminogen (34). The increase in uPA activity observed in macrophage-conditioned media following incubation with laminin was not due to enhanced kinetics of plasminogen activation. In contrast to tPA-mediated plasminogen activation, intact matrices and purified laminin had little or no effect on uPA-mediated plasminogen activation (59, 60). Furthermore, as demonstrated in these studies, incubation of macrophages with laminin resulted in a rapid and sustained increase in the steady state levels of uPA mRNA which paralleled the appearance of uPA activity in their conditioned media.
Laminin is a large (~900 kDa) multidomain heterotrimer consisting of
,
, and
chains (45, 49). The best understood laminin isoform
is derived from Engelbreth Holm Swarm sarcoma matrices (i.e.
Matrigel) and is termed laminin 1 (45, 49). In these studies, we have
utilized synthetic peptides and specific anti-integrin antibodies to
identify the laminin domain and macrophage receptor that mediate the
observed regulation of uPA expression. P1 is a pepsin-generated
fragment of laminin containing portions of the
,
, and
chains
of the short arms (45). The P1 fragment promotes cell attachment and
MMP-9 expression by tumor cells (51, 61, 62). Cellular binding to the
P1 fragment is blocked by antibodies to integrin
1 and
3 subunits
and RGD peptides (51). Laminin peptides CDPYIGSR and YIGSR, derived
from the
chain in the P1 fragment (45), promote cell attachment and
spreading (46, 47). These laminin peptides stimulated MMP-9 expression by tumor cells (48) but block angiogenesis and tumor metastasis (63-65). Incubation of RAW264.7 macrophages with either CDPYIGSR or
YIGSR had no effect on their expression of either uPA or MMP-9.
E8 is an elastase-generated fragment of laminin containing portions of
the ,
, and
chains of the long arm (45). The E8 fragment
promotes cell attachment (51, 61). Cellular binding to the E8 fragment
is blocked by antibodies to either subunit of the
6
1 integrin
(50, 51). The laminin peptide SIKVAV is derived from the
chain of
the E8 fragment (45). SIKVAV peptide promotes tumor invasion,
metastasis, angiogenesis, and MMP expression (30, 48, 66). As reported
here, incubation of RAW264.7 macrophages with SIKVAV also strongly
up-regulated uPA expression. Taken together, these data suggest that
laminin's effect on macrophage uPA expression is mediated through the
binding of the long arm of laminin to the
6
1 laminin
receptor.
To test the hypothesis that the 6
1 integrin mediates matrix
induction of macrophage uPA expression, human THP-1 monocytes were
incubated with a monoclonal antibody directed against integrin
6
subunit (CDw49f) prior to plating on intact matrix. As a control, cells
were incubated with a monoclonal antibody directed against Fc
RIII
(CD16). The adherence of THP-1 monocytes to matrix-coated plastic was
unaffected by either anti-
6 IgG or anti-CD16 IgG. Matrix induction
of THP-1 uPA expression was inhibited by anti-
6 IgG, whereas
anti-CD16 IgG had little or no effect at similar concentrations. In
contrast to uPA, matrix induction of macrophage MMP-9 expression was
unaffected by anti-
6 IgG.
The expression of matrix-degrading proteases regulates a variety of macrophage functions including extravasation, migration, and tissue remodeling (4, 40-42, 67). We and others (3, 4) have demonstrated that macrophage matrix degradation is dramatically enhanced in the presence of plasminogen. Following its activation by uPA, plasmin can degrade several matrix components (6, 7). Matrix degradation can lead to the release and/or activation of matrix-bound growth factors (10-12, 64), which can profoundly influence the cellular expression of plasminogen activators and inhibitors. Finally, plasmin appears to play a role in the proteolytic activation of pro-metalloproteinases (8, 11, 68-70). Results of experiments reported here demonstrate that macrophage binding to laminin is an important stimulus for matrix degradation via stimulation of uPA and MMP-9 expression.