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(Journal of Leukocyte Biology. 2000;68:737-747.)
© 2000 by Society for Leukocyte Biology

Fibronectin-bound TNF-{alpha} stimulates monocyte matrix metalloproteinase-9 expression and regulates chemotaxis

Gayle G. Vaday*, Rami Hershkoviz{dagger}, Michal A. Rahat{ddagger}, Nitza Lahat{ddagger}, Liora Cahalon* and Ofer Lider*

* Department of Immunology, The Weizmann Institute of Science, Rehovot;
{dagger} Department of Internal Medicine, Meir Hospital, Kfar Saba; and
{ddagger} Immunology Research Unit, Lady Davis Carmel Medical Center, Haifa, Israel

Correspondence: Ofer Lider, Ph.D., Dept. of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: ofer.lider{at}weizmann.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor {alpha} (TNF-{alpha}) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF-{alpha} avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF-{alpha} complexing to insoluble ECM components may serve to concentrate its activities to distinct inflamed sites. Herein, we explored the bioactivity and possible function of ECM-bound TNF-{alpha} by examining its effects on MMP-9 secretion by monocytes. Immunofluorescent staining indicated that LPS-activated monocytes deposited newly synthesized TNF-{alpha} into ECM-FN. FN-bound TNF-{alpha} (FN/TNF-{alpha}) significantly up-regulated MMP-9 expression and secretion by the human monocytic cell line MonoMac-6 and peripheral blood monocytes. Such secretion could be inhibited by antibodies that block TNF-{alpha} activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Chemotaxis through ECM gels in the presence of soluble or bound TNF-{alpha} was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac-6 cells to FN/TNF-{alpha} required functional activated ß1 integrins, FN/TNF-{alpha}-induced MMP-9 secretion was independent of binding to ß1 integrins, since MMP-9 secretion was unaffected by: (1) neutralizing mAb to {alpha}4, {alpha}5, and ß1 subunits, which blocked cell adhesion; (2) a mAb that stimulated ß1 integrin-mediated adhesion; and (3) binding TNF-{alpha} to the 30-kDa amino-terminal fragment of FN, which lacks the major cell adhesive binding sites. Thus, in addition to their cell-adhesive roles, ECM glycoproteins, such as FN, may play a pivotal role in presenting proinflammatory cytokines to leukocytes within the actual inflamed tissue, thereby affecting their capacities to secrete ECM-degrading enzymes. These TNF-{alpha}-ECM interactions may serve to limit the cytokine’s availability and bioactivity to target areas of inflammation.

Key Words: inflammation • gelatinase • extracellular matrix


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte transmigration from vascular compartments, across tissue barriers, and through extracellular matrices is a key event during inflammation. The extracellular matrix (ECM) is an insoluble network composed of adhesive glycoproteins, collagens, and proteoglycans, which acts as a scaffold between tissues and modulates cellular functions such as migration and proliferation [1 ]. Various molecules that influence inflammation, including growth factors [2 , 3 ], cytokines, and chemokines [4 5 6 ], and degradative enzymes [7 ], have been shown to interact with ECM moieties. Thus, the molecules encountered by leukocytes in the ECM microenvironment likely provide critical signals that direct and modulate cell functions as needed, such as the synthesis of ECM-modifying enzymes to facilitate cell navigation.

Matrix metalloproteinases (MMPs) are a major family of zinc-containing endo-proteinases involved in degrading ECM components. Several cell types express MMPs during normal and pathological tissue remodeling, and the functions of these enzymes vary according to substrate specificity and inducibility [8 , 9 ]. Although immune cells are not the primary cells responsible for maintaining and organizing connective tissue integrity, their secretion of MMPs likely serves in more specialized roles in inflammation [10 ]. MMP expression by immune cells is greatly modulated by inflammatory mediators, such as tumor necrosis factor {alpha} (TNF-{alpha}, IL-1ß, and IL-4 [9 , 11 ]. Growing evidence also suggests that the same cytokines that influence metalloproteinase expression not only associate with ECM components, but are also subject to enzymatic processing by these enzymes [12 13 14 ].

Previously, our laboratory characterized the biochemical interaction between TNF-{alpha} and fibronectin (FN). TNF-{alpha} binds avidly to FN, specifically to the 30-kDa amino-terminal domain, which lacks the major cell adhesive epitopes. Such FN-bound TNF-{alpha} augments the ß1 integrin-dependent adhesion of CD4+ T cells to the glycoprotein [4 , 15 ]. Similar effects in promoting T cell adhesion were also found in laminin-TNF-{alpha} interactions [16 ]. Collectively, these findings suggest that TNF-{alpha} may serve not only in activating immune cells in circulation, but also in modulating their behavior when cells are in the context of an inflamed ECM microenvironment. However, it was unknown whether FN-bound TNF-{alpha} affects the adhesion of other leukocytes, and whether other physiological activities are affected.

Recent studies have described the capacity of TNF-{alpha} to selectively induce expression of MMP-9 (gelatinase B; 92-kDa gelatinase) by human monocytes [17 ] and monocyte-derived macrophages [18 ]. Furthermore, MMP-9 expression, which is induced during monocyte differentiation into macrophages [19 ], was recently shown to involve an autocrine loop of TNF-{alpha} that enhances {alpha}5ß1 integrin expression levels [20 ] and requires FN-mediated cell adhesion [21 ]. Considering our previous findings that TNF-{alpha} binds to FN, we speculated that this interaction may potentiate changes in monocyte behavior, particularly adhesion and MMP secretion, via cooperative integrin- and TNF receptor-binding.

Herein, we pursued a study to determine the role of ECM adhesive ligands in regulating MMP-9 expression and secretion by presenting bound TNF-{alpha} to monocytes. Our study examined the bioactivity of FN-bound TNF-{alpha} by analyzing its effects on MMP-9 secretion by human peripheral blood monocytes and the nonadherent monocytic cell line MonoMac-6, which was established from human monoblastic leukemia cells [22 ]. We further examined the effects of FN-bound TNF-{alpha} on MonoMac-6 cell migration through ECM gels. The results demonstrate that FN-bound TNF-{alpha} induces MMP-9 synthesis in a mechanism independent of ß1 integrin-mediated cell adhesion. We also found that MMPs are of critical importance during cell migration through ECM containing TNF-{alpha} bound to FN. These results suggest that ECM components, such as FN, likely serve a significant biological role in concentrating and presenting cytokines found at elevated levels at inflamed sites to leukocytes, thereby providing combinations of signals during their navigation into tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Gelatin A, lipopolysaccharide (LPS), ECM gel, GRGDS and RGES peptides, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). All materials and equipment for electrophoresis were purchased from Bio-Rad (Hercules, CA) unless indicated otherwise. Anti-human MMP-2 mAb was from Calbiochem (La Jolla, CA), anti-human MMP-9 mAb and anti-human CCR2 mAb were from R & D (Minneapolis, MN), and human plasma FN was from Chemicon (Temecula, CA). Coverslips covered with endogenous ECM made by bovine corneal endothelial cells were purchased from Novamed (Jerusalem, Israel). The 30-kDa amino-terminal fragment of FN was obtained from Dr. Kenneth Yamada (National Institutes of Health). Recombinant human TNF-{alpha} was kindly donated by Dr. Yehuda Chowers (Tel Hashomer Medical Center, Israel). MonoMac-6 cells were donated by Peptor Laboratories (Nes Ziona, Israel), and mAb 8A2 was a gift from Dr. J. M. Harlan (University of Washington). Neutralizing mAb to TNF-{alpha} and to TNF RI and TNF RII were purchased from PharMingen (San Diego, CA) and R & D, respectively. A hydroxamic acid inhibitor of MMPs, GM6001 (Galardin [23 ]), was donated by Dr. Benny Shilo (Weizmann Institute of Science). Monoclonal antibodies directed to human integrin subunits {alpha}4, {alpha}5, {alpha}6, and ß1 were from Serotec (Oxford, UK).

Cells and cell culture conditions
Long-term cultures of MonoMac-6 cells were maintained in RPMI medium containing HEPES (Life Technologies), supplemented with L-glutamine (200 mmol/L; Merck), penicillin (100 U/mL)-streptomycin (100 µg/mL; Beit Haemek, Israel), bovine insulin (2.5 µg/mL; Sigma), sodium pyruvate (200 mmol/L; Merck), and 10% fetal calf serum (Beit Haemek, Israel). Culture plates were coated with human FN (5 µg/mL) in phosphate-buffered saline (PBS) at 4°C overnight. TNF-{alpha} (10 ng/mL to 1 µg/mL) binding to FN was done in PBS for 2 h at 37°C, then the plates were blocked for 20 min with serum-free AIM-V media containing the same supplements (Life Technologies, Gaithersburg, MD). Experiments were performed by washing cells twice and incubating at a density of 1 x 106 cells/mL of AIM-V media. PMA (5 ng/mL), LPS (50 ng/mL), and soluble TNF-{alpha} (sTNF; 1 ng/mL) were used for cell activation and were added at the time of plating. Specific mAb were used to neutralize the bioactivities of TNF-{alpha} (1.5 µg/mL), TNF RI (6 µg/mL), and TNF RII (1.5 µg/mL) at concentrations reported by the manufacturer to inhibit approximately 50% lysis of the TNF-{alpha}-sensitive cell line L929. Specific neutralizing mAb to {alpha}4, {alpha}5, ß1, and {alpha}6 integrin subunits (5 µg/mL), ß1 integrin affinity-modulating mAb 8A2 (4 µg/mL), and GRGDS and RGES peptides (100 µg/mL) were used in a cell adhesion assay. Cells were preincubated for 30 min on ice with the mAb or peptides before plating. Supernatants were stored immediately at -70°C.

Peripheral blood monocytes were isolated from healthy donors according to Treves et al. [24 ] by fractionating buffy coats on Ficoll-Hypaque gradients (Novamed, Israel). Mononuclear cell fractions were washed extensively in Ca2+-Mg2+-free PBS and further separated by adhesion to plastic in culture flasks at 5 x 106 cells/mL in RPMI containing L-glutamine, penicillin-streptomycin, and 2% newborn calf serum (Beit Haemek) for 2 h at 37°C. Nonadherent cells were removed, and adherent cells were washed extensively with warm Earle’s balanced salt solution (EBSS). Adherent cells were then cultured for 16 h at 37°C in complete RPMI containing 10% newborn calf serum, which resulted in the majority of cells being loosely adherent and easily detached by pipetting with cold EBSS. The purity of monocytes was verified to be >=85% by FACS analysis using fluorescein isothiocyanate (FITC)-conjugated anti-CD14 mAb (Immunoquality Products, The Netherlands). The purification procedure did not activate the monocytes because less than 5% of the overnight culture was found to be IL-2R{alpha} subunit (CD25)-positive, a sensitive marker of cell activation [25 ]. Experiments with peripheral blood monocytes were performed in AIM-V media after purification.

Immunofluorescent staining
MonoMac-6 cells were cultured in complete AIM-V medium at a density of 0.5 x 106 cells/mL in six-well cluster plates and treated with LPS (50 ng/mL) for 2 h. One milliliter of the culture was then transferred to four-well cluster plates containing glass coverslips coated with ECM derived from bovine corneal endothelial cells (Novamed, Israel), and cultured for 2 h. The culture medium was then removed, and TNF-{alpha} secretion was quantitated by sandwich enzyme-linked immunosorbent assay (ELISA) using purified and biotinylated mAb specific for human TNF-{alpha} (PharMingen). The coverslips were washed three times with PBS and fixed in 3.5% formaldehyde for 20 min. After extensive washing and blocking with 2% ovalbumin, double immunostaining was performed using rabbit anti-human FN pAb (10 µg/mL; Calbiochem) and mouse anti-human TNF-{alpha} mAb (10 µg/mL; PharMingen), followed by rhodamine-conjugated anti-rabbit IgG and FITC anti-mouse IgG antibodies, respectively. Stained coverslips were mounted onto glass slides and photographed on Kodak TMAX-3200 film using a Zeiss Axiolab fluorescent microscope.

Gelatin zymography and immunoblotting
Protein concentrations of the supernatants were determined using Bio-Rad Protein Assay reagent. Equal concentrations of protein were analyzed on 10% polyacrylamide-sodium dodecyl sulfate (SDS) gels embedded with 1 mg/mL gelatin A under nonreducing conditions using a Bio-Rad Protean II system. Gels were incubated in 2% Triton X-100 for 1 h to remove SDS and renature the proteins, washed three times with H2O for 5 min each, then incubated at 37°C in 50 mM Tris-HCl pH 7.5 + 5 mM CaCl2 for 22–24 h. Subsequently, gels were stained with Coomassie Blue R-250. Clear bands indicate gelatinolytic activity. Densitometric scanning of gels was performed using a UMAX Astra 1220S scanner and the NIH Image analysis program.

Samples were concentrated 2.5-fold using Millipore Ultrafree-MC units consisting of filters to allow concentration of proteins >=30 kDa (for MMP-2 and MMP-9 analysis). Equal concentrations of proteins were subjected to electrophoresis under reducing conditions on 10% polyacrylamide-SDS gels, then samples were transferred to nitrocellulose for immunoblotting. Monoclonal antibodies to MMP-2 or MMP-9 were used as primary antibodies, horseradish peroxidase-conjugated goat anti-mouse IgG were used as secondary antibodies, and detection was done using Pierce Supersignal reagent (Rockford, IL).

Migration assay
Cell migration was measured with Transwell inserts (6.5-mm diameter; Corning, NY) fitted with polycarbonate filters (5-µm pore size). MonoMac-6 cells were radiolabeled with 51Cr (DuPont-NEN, Boston, MA), then resuspended in RPMI/0.1% BSA containing L-glutamine and antibiotics. FN (0.5 µg/sample) was complexed with TNF-{alpha} (0.5 ng/sample) by incubating for 2 h at 37°C. Replicate suspensions of 105 cells in 0.1 mL were added over filters coated with either FN (25 µg/mL) with or without bound TNF-{alpha} (0.4 ng), or with a continuous even coating of 15 µL of ECM gel (Sigma) supplemented with FN or FN-complexed TNF-{alpha}. The bottom well contained 0.6 mL of medium with or without 50 ng/mL human MCP-1 (Peprotech; Rocky Hill, NJ). The hydroxamic acid compound GM6001, a well-characterized inhibitor of MMPs [23 ], was added to the lower compartment, cell suspension, and the ECM gel at 10 µM. Experiments were terminated after 4-h migration through FN and after 20-h migration through ECM gel by collecting the lower media containing transmigrated cells. These cells were centrifuged and resuspended in 0.1 mL lysis buffer (1 M NaOH/0.1% Triton X-100), and samples were analyzed by gamma counting. Total counts of 0.1 mL of the original cell suspension was also measured, and the percentage of cell migration was quantified. Data are expressed as percent (± SD) of control unstimulated cells or percentage of inhibition by GM6001.

FACS analysis
Cell staining was performed on MonoMac-6 cells exposed to soluble and FN-bound TNF-{alpha} using human mAb anti-CCR2 and an irrelevant control mouse IgG2b followed by FITC-conjugated goat anti-mouse Ab (Jackson Immunoresearch, West Grove, PA) using a FACSort instrument (Becton Dickinson).

Cell adhesion
MonoMac-6 cell adhesion was studied using techniques previously performed with other cell types [4 , 15 ]. Microtiter 96-well culture plates were coated with FN (1 µg/well) in PBS overnight at 4°C. To analyze the effects of FN-associated TNF-{alpha} on cell adhesion, TNF-{alpha} (20 ng/well) was subsequently bound for 2 h at 37°C, then plates were blocked with 0.1% BSA. Cells were radiolabeled with 51Cr, then resuspended in RPMI/0.1% BSA. Pre-incubations with anti-integrin mAb or GRGDS and RGES peptides were done on ice for 30 min before plating the cells at a density of 105 cells per well in 100 µL. PMA (25 ng/mL) or sTNF-{alpha} (1 ng/mL) were added simultaneously with the cells. Plates were incubated at 37°C for 1 h in a humidified 5% CO2 incubator, then washed three times with PBS to remove nonadherent cells. Adherent cells were lysed in 1 M NaOH/0.1% Triton X-100, and lysates were removed for gamma counting. Results are expressed as the mean percent (± SD) of bound MonoMac-6 cells in quadruplicate wells.

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
MonoMac-6 cells were cultured at a density of 1 x 106 cells/mL in six-well culture plates prepared with immobilized FN (5 µg/mL) in the presence of soluble or bound TNF-{alpha} (4 ng/mL) and PMA (5 nM). The cell cultures were collected by centrifugation at various times, supernatants were used in gelatin zymography, and cells were lysed in TriReagentTM (Molecular Probes). Total RNA was extracted according to the manufacturer’s instructions, and 30 µg were treated with DNase I (Amersham Pharmacia, Piscataway, NJ) before analysis by semiquantitative RT-PCR. RNA (1 µg) samples were reverse transcribed at 37°C for 1 h with the use of 200 µM deoxynucleotides (Sigma), 5 µM random hexamers (Amersham Pharmacia), 20 U RNAguard (Amersham Pharmacia), and 20 U/µg of MMLV-RT (US Biochemicals, Cleveland, OH). After synthesis of first-strand cDNA, the samples were boiled for 10 min to inactivate the enzyme. The amplification reaction consisted of 0.4 µM of each primer, 100 µM of dNTPs (Sigma), 0.5 U of Taq polymerase (Roche Molecular Biochemicals, Mannheim, Germany), Flash anti-Taq (Chimrex), and 25–200 ng of the reverse-transcribed RNA, in a final volume of 15 µL. The linear ranges of amplification and cDNA concentrations and optimal time of expression (24 h) were determined for each transcript, and amplification was performed within those linear ranges. Amplification of MMP-9 (200 ng) was conducted by denaturing the cDNA at 94°C for 30 s, annealing at 54°C for 30 s, and elongating at 72°C for 30 s for 33 cycles. Amplification of GAPDH (25 ng) was carried out under the same conditions. PCR samples were subjected to electrophoresis on 1% agarose gels and analyzed by densitometric scanning (Bio-Imaging system, Dinco-Renium, Jerusalem, Israel) using TINA software (Raytest, Straubenhardt, Germany). The oligonucleotide sequences used were: MMP-9 sense, 5’-GGCCCTTCTACGGCCACT; MMP-9 antisense, 5’-CAGAGAATCGCCAGTACTT; GAPDH sense, 5’-ACCACAAGTCCAATGCCATCAC; and GAPDH antisense, 5’-TCCACCACCCTGTTGCTGTA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} associates with matrix-FN
Our laboratory previously demonstrated that purified TNF-{alpha} binds with high avidity to FN [4 ]. We sought to establish this interaction between FN and TNF-{alpha} as a model to study the effects of ECM-bound cytokines on MMP secretion from monocytes. Deposition of TNF-{alpha} secreted by activated MonoMac-6 cells into ECM was examined by incubating LPS-treated cells with their conditioned media on glass coverslips covered with an intact ECM. Both FN and TNF-{alpha} were examined using indirect immunofluorescent double staining. Thick fibrils of FN were found throughout the ECM (Fig. 1A and C ), and TNF-{alpha} was deposited in small punctate patterns at specific sites of colocalization along these strands of FN (Fig. 1B) . Such TNF-{alpha} was derived from LPS-activated monocytes and did not originate from the source of the ECM because TNF-{alpha} was not detected in ECM alone (Fig. 1D) ; control staining with an irrelevant antibody resulted in similar negative detection. Measurement of TNF-{alpha} in the conditioned medium by ELISA indicated that 0.1 ng/mL of TNF-{alpha} was secreted by the activated monocytes (data not shown). These findings proved that TNF-{alpha} secreted by LPS-activated monocytes is consequently deposited and complexed into native ECM. Furthermore, colocalization of FN and TNF-{alpha} suggests that ECM constituents may serve as a depot to sequester and concentrate TNF-{alpha} in distinct sites for presentation to immune cells.



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  		Figure 1. Colocalization of ECM FN and TNF-{alpha} secreted by activated monocytes. To examine whether TNF-{alpha} synthesized by monocytes may be deposited and complexed into ECM, MonoMac-6 cells were treated with LPS for 2 h, then the culture was transferred and incubated for 2 h in wells containing ECM-coated glass coverslips (A, B). Controls were incubated with medium only (C, D). After incubation for 2 h, the coverslips were fixed and double stained using rabbit anti-human FN pAb and mouse anti-human TNF-{alpha} mAb, followed by rhodamine-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG antibodies, respectively. Two photographs were taken of the same field using different light filters to visualize rhodamine and FITC staining at x400 magnification. (A, C) FN staining in intact ECM; (B) TNF-{alpha} staining in ECM incubated with monocytes; and (D) TNF-{alpha} staining in ECM control. The bar in the lower panel represents 35 µm.

 
FN-complexed TNF-{alpha} stimulates MMP-9 secretion
We pursued a further examination of the biological effects of ECM-immobilized TNF-{alpha}, which immune cells may encounter in inflamed sites, by studying its effect on MMP secretion from monocytes. Specifically, the bioactivity of ECM-bound TNF-{alpha} in stimulating MonoMac-6 production of MMP-9 was explored, using FN as a representative ECM protein. Binding assays were performed using iodinated TNF-{alpha} to quantitate the amount of TNF-{alpha} that binds to FN. Approximately 4% of the added TNF-{alpha} remained bound to FN after extensive washing, and the majority remained bound after incubation with cells overnight (data not shown).

Next, we studied the effects of matrix-complexed TNF-{alpha} on MMP expression by monocytes. Cells were incubated for 24 h in 24-well culture plates on plastic alone, FN, or FN with increasing concentrations (0.4–40 ng/mL) of FN-bound TNF-{alpha} or sTNF-{alpha}; then supernatants were analyzed by gelatin zymography to determine MMP-9 secretion (Fig. 2A ). MMP-9 secretion was below the level of detection in control untreated cells incubated on plastic or FN alone. Treatment with soluble TNF-{alpha} resulted in a significant increase in MMP-9 secretion, as previously demonstrated in other monocyte cell lines [20 , 21 ]. Incubation on FN with bound TNF-{alpha} also caused a marked increase in MMP-9 secretion, and this response was augmented with corresponding increases in concentrations of bound TNF-{alpha} (Fig. 2A) . The dose response to sTNF-{alpha} and FN/TNF-{alpha} on induction of MMP-9 secretion was examined by densitometric scanning of MMP-9-mediated degradation on the gelatin zymograms (Fig. 2B) . Although the sTNF-{alpha} induced MMP-9 secretion at appreciably lower concentrations than FN/TNF-{alpha}, in parallel, both forms of the cytokine demonstrated a dose-dependent increase of MMP-9 secretion. All subsequent experiments were performed using the bound TNF-{alpha} concentration of 4 ng/mL and the sTNF-{alpha} concentration of 1 ng/mL, since these concentrations were within the peak of induction of MMP-9.



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  		Figure 2. Dose response to FN-bound and soluble TNF-{alpha}. MonoMac-6 cells were exposed to increasing amounts of either soluble or FN-bound TNF-{alpha} (0.4–40 ng/mL) for 24 h. Supernatants were analyzed by gelatin zymography on 10% polyacrylamide-SDS gels under nonreducing conditions. (A) Gelatinase activity was seen as clear degraded bands migrating to 92 kDa (MMP-9) and 72 kDa (MMP-2). (B) Densitometric scanning of MMP-9 on gelatin zymograms demonstrating the dose response to soluble or FN-bound TNF-{alpha}. Data are presented as the ratio over the control (no TNF-{alpha}). Representative of three separate experiments.

 
To determine the time course of induction of MMP-9 expression, cells were exposed to plastic, FN, or FN/TNF-{alpha} for 3, 6, 24, or 48 h. Supernatants were collected and analyzed by zymography (Fig. 3A ) and immunoblotting (Fig. 3B) , and cells were lysed for RNA isolation to determine MMP-9 gene regulation (Fig. 3C) . The results obtained by zymography indicated that MMP-2 was secreted after 24 h and continued until 48 h, regardless of the culture treatments (Fig. 3A) . MMP-9 was also secreted after 24 h, but only in cells exposed to FN-bound TNF-{alpha} or sTNF-{alpha}. Gelatinases in the supernatants from the 24-h time point were further identified using anti-MMP-9 and anti-MMP-2 mAb. Analysis by the Western blotting indicated that MMP-2 was secreted constitutively in all conditions, and MMP-9 secretion was only detected in supernatants of cells exposed to FN-bound TNF-{alpha} or sTNF-{alpha} (Fig. 3B) , thus corroborating with the results found by zymography (Fig. 3A) . To determine whether such up-regulation of MMP-9 production was regulated at the transcriptional level, semiquantitative RT-PCR was performed. The time course of expression and the linear ranges of RNA concentrations and amplification for MMP-9 and GAPDH to be used in each reverse transcription and PCR reaction were predetermined (data not shown). We found that MMP-9 was regulated at the transcriptional level (Fig. 3C) , wherein MMP-9 expression was up-regulated in cells exposed to soluble and bound TNF-{alpha}.



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  		Figure 3. Time course of expression and secretion of MMP-9 and MMP-2. MonoMac-6 cells were exposed to plastic, FN, or FN-bound TNF-{alpha} for 3, 6, 24, and 48 h in six-well cluster plates at a density of 1 x 106 cells/mL. Supernatants were collected for protein analyses, and total RNA was isolated from the cells to determine gene expression. (A) The time course of MMP-9 and MMP-2 secretion was determined by gelatin zymography. (B) Supernatants from the 24-h time point were further analyzed by concentrating the proteins and performing Western blots with mAb against human MMP-9 and MMP-2. (C) RNA was isolated from the cells and analyzed by semi-quantitative RT-PCR. The linear ranges for RNA concentrations and amplification cycles for MMP-9 and GAPDH were determined. Amplification was performed within the linear ranges for both MMP-9 and GAPDH, and the samples were run on 1% agarose gels for analysis by densitometric scanning. Lane 1, control untreated; lane 2, FN; lane 3, FN/TNF; lane 4, FN + sTNF. Data for MMP-9 were normalized to GAPDH. The results are presented as fold increase from control untreated cells ± SD and are representative of three separate experiments.

 
Role of TNF-{alpha} receptors in MMP-9 secretion
TNF-{alpha} binding to cells is mediated via two membrane receptors, designated TNF RI (p55) and TNF RII (p75), which transmit differential intracellular signals [26 , 27 ]. Both receptors are highly expressed (>85% of the population) on resting MonoMac-6 cells, as determined by flow cytometry (data not shown). To determine which receptor plays a role in mediating TNF-{alpha}-induced MMP-9 secretion, specific antibodies were used to neutralize the cytokine, as well as its receptors TNF RI and TNF RII. Cells were preincubated with the antibodies on ice, then incubated overnight with soluble or bound TNF-{alpha}, and secretion was measured by zymography. The cytokine’s activities were clearly blocked by incubation with TNF-{alpha}-specific antibodies. It is interesting that neutralizing both receptors TNF RI and TNF RII caused marked reductions in MMP-9 secretion in cells incubated on FN/TNF-{alpha}, indicating that both molecules are involved in TNF-{alpha}-induced MMP-9 (Fig. 4 ).



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  		Figure 4. Neutralization of TNF-{alpha} and its receptors TNF RI (p55) and TNF RII (p75). To block the bioactivities of TNF-{alpha} and its receptors, MonoMac-6 cells were pre-incubated with neutralizing mAb specific for TNF-{alpha}, TNF RI, and TNF RII. Cells were cultured on FN alone or FN/TNF-{alpha}, or with sTNF-{alpha} on FN for 24 h, and supernatants were analyzed by gelatin zymography. Lane 1, no antibody added; lane 2, plus anti-TNF-{alpha} mAb (1.5 µg/mL); lane 3, plus anti-TNF RI (6 µg/mL); lane 4, plus anti-TNF RII (1.5 µg/mL). The gel shown is representative of three separate experiments.

 
Role of integrins in monocyte adhesion
Cell adhesion molecules play a significant role in mediating cell attachment to ECM ligands during leukocyte extravasation. Several studies have demonstrated that leukocyte integrins and adhesion molecules also influence immune cell extravasation by modulating MMP secretion [28 29 30 31 32 ]. Therefore, we investigated the role of FN-specific ß1 integrins in MonoMac-6 cell adhesion to FN, as well as MMP-9 secretion. First, we analyzed the role of such receptors in MonoMac-6 cell adhesion to FN [4 , 15 ]. Less than 10% cell adhesion was observed in cells incubated on FN with or without sTNF-{alpha}, as well as on FN/TNF-{alpha}. However, while the addition of PMA significantly increased cell adhesion on FN, no remarkable differences in adhesion were observed in the presence of soluble or bound TNF-{alpha} (Fig. 5A [15 , 33 ]). Thus, we conclude that neither soluble nor FN-bound TNF-{alpha} play a significant role in augmenting ß1 integrin-mediated monocyte adhesion to FN.



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  		Figure 5. Integrin-mediated cell adhesion to FN and FN/TNF-{alpha}. Cell adhesion assays were performed to determine the role of ß1 integrins in mediating MonoMac-6 cell adhesion to FN and FN/TNF-{alpha}. Cells were 51Cr-radiolabeled, then incubated for 1 h at 37°C on microtiter wells pre-coated with FN or FN/TNF-{alpha}. Treatments with mAb consisted of pre-incubations for 30 min on ice before plating the cells and the addition of PMA or sTNF-{alpha}. (A) Monocyte adherence on FN or FN/TNF-{alpha} with or without PMA treatment. (B) Cell adherence with PMA treatment in the presence of blocking mAb (5 µg/mL) directed to {alpha}4, {alpha}5, ß1 integrin subunits, or GRGDS or RGES peptides (100 µg/mL). (C) Cell adhesion to FN or FN/TNF-{alpha} with or without integrin affinity-modulating mAb 8A2 (4 µg/mL) [34 ]. Plates were washed extensively to remove unbound cells, bound cells were lysed, and the resultant supernatants were removed and analyzed by gamma counting. For each experimental group, the results are expressed as the mean percentage ± SD of bound MonoMac-6 cells from quadruplicate wells.

 
To further support this notion, cell adhesion assays were performed using neutralizing antibodies directed to integrin subunits, which are important in adhesion to FN. Antibodies specific for FN-binding integrin subunits, i.e. {alpha}4, {alpha}5, and the common ß1 chain, significantly reduced MonoMac-6 cell adhesion to FN, regardless of treatment with soluble or bound TNF-{alpha}. However, mAb directed against the integrin subunit {alpha}6, of which the {alpha}6ß1 integrin specifically binds laminin, did not alter cell adherence to FN in the presence of FN/TNF-{alpha} or sTNF-{alpha} (Fig. 5B) . Similar results of reduced adhesion were observed with GRGDS peptides, which contain the cell-adhesive Arg-Gly-Asp sequence, but not with control RGES peptides (Fig. 5B) .

Cell adhesion assays were also performed with the ß1 integrin affinity-enhancing mAb 8A2, which has been shown to enhance adherence of leukocytes to ECM components, as well as endothelial cells [34 ]. Indeed, we found that mAb 8A2 augmented monocyte adhesion to both FN and FN/TNF-{alpha} (Fig. 5C) . Taken together, these results suggest that FN-specific ß1 integrins play a significant role in mediating MonoMac-6 cell adhesion to FN/TNF-{alpha}; however, stimulation with TNF-{alpha} is not likely involved in this process.

Role of integrins in MMP-9 secretion
It was unknown whether induction of MMP-9 in cells incubated on FN/TNF-{alpha} involved cooperative signaling between FN-ß1 integrin binding and TNF-TNF receptor binding. One characteristic of the FN/TNF-{alpha} complex that distinguishes it from sTNF-{alpha} treatment on FN substrates is that both molecules are proximal when encountered by the cells. It was evident that both TNF-{alpha} receptors were engaging with FN/TNF-{alpha} to induce MMP-9 secretion, but not adhesion to FN. Furthermore, it was clear that {alpha}5ß1 and {alpha}4ß1 integrins were engaging with FN to mediate adhesion. However, it was unknown whether integrin and TNF-{alpha} receptors are both involved, possibly through a specific crosstalk, in up-regulating MMP-9 secretion upon exposure to FN/TNF-{alpha}.

To determine the possible role of FN-ß1 integrin binding in FN/TNF-{alpha}-induced MMP-9 expression, cells were incubated with specific blocking or affinity-modulating mAb. These mAbs were used at the same concentrations that either blocked (anti-{alpha}4, {alpha}5, ß1 mAb) or induced (8A2 mAb) cell adhesion. Incubations with anti-{alpha}4, -{alpha}5, -ß1, and -{alpha}6 mAbs, as well as with mAb 8A2, were performed for 24 h, and the supernatants were analyzed by zymography. Surprisingly, although treatment with mAb 8A2 stimulated ß1 integrin-mediated adhesion to FN (Fig. 5C) , the antibody did not stimulate MMP-9 secretion from cells plated on FN/TNF-{alpha} (Fig. 6A ). The mAb 8A2 also did not enhance MMP-9 secretion from cells treated with sTNF-{alpha} (Fig. 6A) . Similarly, the mAbs (anti-{alpha}4, -{alpha}5, -ß1) that inhibited PMA-induced adhesion to FN and FN/TNF-{alpha} did not alter PMA- or TNF-{alpha}-induced MMP-9 secretion because no detectable differences in MMP-9 secretion were found in cultures treated with these blocking mAb and control cells (anti-{alpha}6 or none; Fig. 6B ). These results further suggest that FN-binding ß1 integrins do not mediate MMP-9 synthesis.



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  		Figure 6. Effects of anti-integrin blocking or stimulating antibodies and FN binding sites on MMP-9 secretion. MonoMac-6 cells were pre-incubated with (A) ß1 integrin stimulating mAb 8A2 (4 µg/mL) or (B) blocking mAb anti {alpha}4, {alpha}5, or ß1 (5 µg/mL) for 30 min on ice, then plated onto the various substrates with or without sTNF-{alpha} treatment. (C) TNF-{alpha} was bound to intact FN or the 30-kDa amino-terminal fragment of FN (FN-N’). MonoMac-6 cells were plated onto either intact FN (5 µg/mL) or FN-N’ (5 µg/mL) alone, or FN/TNF-{alpha} or FN-N’/TNF-{alpha} for 24 h. Supernatants were subjected to gelatin zymography to examine MMP-9 secretion. The gels shown are representative of three separate experiments.

 
If indeed FN-specific ß1 integrins do not play a role in the FN-complexed TNF-{alpha} effect on MMP-9 secretion by monocytes, then FN may serve to anchor TNF-{alpha}, and perhaps present it to TNF-{alpha} receptors. To further support this notion, TNF-{alpha} was complexed to FN sites remote from the integrin-binding domains. TNF-{alpha} was bound to the amino-terminal 30-kDa fragment of FN (FN-N’), which lacks the Arg-Gly-Asp ({alpha}5ß1-binding) and Leu-Asp-Val ({alpha}4ß1-binding) domains, at a site distinct from the fibrin- and heparin-binding sites. This FN-N’ fragment was previously characterized to specifically bind TNF-{alpha} with high affinity at slow dissociation rates. Furthermore, it was demonstrated that FN-N’-bound TNF-{alpha} does not support cell adhesion [4 ]. In this study, we found that FN-N’-bound TNF-{alpha} markedly increased MMP-9 secretion (Fig. 6C) . Densitometric scanning also indicated a >=30% increase in secretion compared to intact FN/TNF-{alpha} (data not shown), which may be explained by a higher concentration of TNF-{alpha} binding sites on plates coated with FN-N’ compared with intact FN. These findings provide evidence that integrin-mediated adhesion to FN/TNF-{alpha} and FN/TNF-{alpha}-induced MMP-9 synthesis are each independent processes.

To confirm that FN/TNF-{alpha}-induced MMP-9 is also secreted by human peripheral monocytic cells in an integrin-independent mechanism, we further studied these characteristics in cells isolated from healthy human donors. Zymograms of cell supernatants from different donors indicated that both FN-bound and FN-N’-bound TNF-{alpha} augments secretion of MMP-9 (Fig. 7 ). Thus, FN binding to cell surface integrins on MonoMac-6 cells, as well as peripheral blood monocytes, apparently does not mediate the MMP-9-inducing effects of FN-bound TNF-{alpha}. These findings provide evidence that, although FN supports activated monocyte adhesion via integrin-binding, FN complexed to TNF-{alpha} likely serves as a support to localize and concentrate the cytokine and induce MMP-9 synthesis independent of such adhesion.



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  		Figure 7. Effects of FN/TNF-{alpha} and FN-N’-TNF-{alpha} on MMP-9 secretion from peripheral blood monocytes. Monocytes were isolated from the peripheral blood of three healthy donors. Plates were coated with either FN (5 µg/mL) or FN-N’ (5 µg/mL), and bound with TNF-{alpha} (4 ng/mL). Cells were incubated in serum-free AIM-V media on FN/TNF-{alpha} or FN-N’-TNF-{alpha}, or treated with LPS (50 ng/mL) or sTNF-{alpha} (1 ng/mL) on FN for 24 h. Supernatants were analyzed by gelatin zymography, and densitometric scanning was performed to determine MMP-9 secretion from the three donors. Data are presented as the ratio of densitometric units over the units of the control FN sample.

 
Effects of FN-bound TNF-{alpha} in monocyte chemotaxis
MMPs are implicated in the ECM degradation required for migration of monocytes [35 , 36 ], as well as other leukocytes [30 ]. Thus, it was pertinent for us to investigate the effects of MMP-9 synthesis, which is up-regulated by FN-bound TNF-{alpha}, on MonoMac-6 cell chemotaxis toward the chemokine MCP-1. Migration assays were performed using Transwell chambers housed with filters coated with FN, FN with bound TNF-{alpha}, or ECM gels. Migration was assessed by collecting and counting the cells that transmigrated into the lower well. It is interesting that we found that while MonoMac-6 cells were stimulated to migrate through FN-coated filters toward MCP-1 after 3 h, the presence of either soluble TNF-{alpha} or FN/TNF-{alpha} inhibited such chemotaxis (Fig. 8A ; P < 0.05).



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  		Figure 8. Effects of TNF-{alpha} on MonoMac-6 cell chemotaxis. Migration assays were performed using Transwell inserts containing polycarbonate filters. Experiments were performed using radiolabeled cells in the upper compartment with or without soluble TNF-{alpha} (0.5 ng/sample), and human MCP-1 (50 ng/mL) was placed in the lower compartment for chemotaxis. Where indicated, the MMP inhibitor GM6001 was added to the upper and lower media, as well as in the ECM gel. FN and TNF-{alpha} were complexed before coating the filters or supplementing ECM gels. (A) Migration through FN and FN/TNF-{alpha} was measured after 3 h, and (B) migration through ECM gels was measured after 20 h. (C) The inhibitory effects of GM6001 (10 µm) on cell migration was determined for cells migrating through ECM gels. Transmigrated cells in the bottom chamber were lysed and analyzed in a gamma counter. Data are presented as percentage of control unstimulated cells or percentage of inhibition, and is representative of three separate experiments.

 
To study both the effects of FN/TNF-{alpha} in mediating MonoMac-6 cell chemotaxis and the putative role of MMP-9 in this process, migration assays through ECM gels supplemented with FN, with or without pre-bound TNF-{alpha}, were performed. Assays were done in the absence (Fig. 8B) or presence (Fig. 8C) of a specific inhibitor of MMPs [23 ]. The hydroxamic acid compound GM6001 has been extensively characterized for its ability to specifically inhibit metalloproteinase activity [23 , 30 ]. GM6001 was added to the upper chambers containing the cells, in the lower wells containing the chemokine, and in the ECM gel coating the filters. After 20 h, transmigrated cells in the lower wells were collected, and cell migration was measured and presented as percentage over control unstimulated cells. Our findings indicated that TNF-{alpha} had no apparent effect in enhancing MCP-1-induced chemotaxis through the ECM gel after 20 h (Fig. 8B) , which is the time required for significant MMP-9 secretion. In fact, an increase in migration toward MCP-1 was found in ECM gels containing FN/TNF-{alpha} (P < 0.05). However, the addition of the MMP inhibitor GM6001 resulted in appreciable decreases in cell migration toward MCP-1 (P < 0.05) in the presence of soluble or bound TNF-{alpha} compared to MCP-1 alone (Fig. 8C) . To determine whether the levels of the MCP-1 receptor, CCR2, were affected after 3 or 20 h of exposures to soluble or bound TNF-{alpha}, cells were stained with anti-CCR2 antibodies and analyzed by flow cytometry. Results showed that TNF-{alpha}-treated cells expressed CCR2 levels similar to those of control cells after 3 h, whereas a slight reduction (17 ± 2.5%) in CCR2 expression was observed after exposure to TNF-{alpha} for 20 h (data not shown). Collectively, these findings suggest that monocyte chemotaxis may be differentially regulated according to the deposition of TNF-{alpha} in ECM, wherein such deposition may lead to a rapid, transient signal to stop at the site, via an as yet unidentified mechanism. Although the TNF-{alpha} signal may reduce CCR2 expression after prolonged exposure, it may also lead to a concomitant shift toward MMP-mediated migration in inflamed tissues.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Inflammation is marked by the release of a variety of cytokines and modifying enzymes, and their presence within inflamed tissues likely involves dynamic interactions with components of the ECM microenvironment [37 38 39 ]. The targeting of cytokines to specific sites of inflamed tissues, as well as the avidity and duration of their binding to ECM constituents, may be significant factors in the cellular response to inflammation [39 ]. Previous studies by our laboratory [4 5 6 ] and others [2 , 3 , 40 ] have identified cytokines that not only interact with ECM moieties, but also modulate the behavior of immune cells in this context. For example, FN-bound TNF-{alpha} enhances T lymphocyte adhesion via ß1 integrin binding [4 , 15 ]. Immune cell extravasation also requires the degradation of ECM components and debridement of the inflamed site by MMPs and other proteases. Because activated monocytes are the primary source of TNF-{alpha}, and cytokine release may occur within the ECM, we postulated that ECM-bound TNF-{alpha} may influence the secretion of MMP-9 by monocytes. Our results indicate that TNF-{alpha} released by LPS-activated monocytes indeed binds to matrix FN, and that FN-bound TNF-{alpha} induces the secretion of MMP-9 via TNF RI and TNF RII. Chemotaxis through ECM gels complexed with TNF-{alpha} was inhibited by a specific MMP inhibitor. Moreover, unlike their effects on monocyte adhesion to FN, ß1 integrins do not mediate FN-complexed TNF-{alpha} induction of MMP-9 secretion.

Leukocyte interactions with ECM constituents are mediated by binding between adhesion molecules expressed on the cell membrane and specific ligands in the matrix [1 , 42 ]. TNF-{alpha} binding to FN presents a unique complex because the stimulatory effects of the cytokine are immobilized onto a matrix component with strong pro-adhesive properties. FN major cell adhesive domains include the Arg-Gly-Asp central domain, which binds several integrins, particularly the "classical" FN receptor {alpha}5ß1, and the COOH-terminal variable region, which contains the {alpha}4ß1 integrin binding site Leu-Asp-Val [32 , 41 42 43 ]. Hence, determination of the role of monocyte integrins, particularly those composed of the ß1 subunit, in mediating not only cell adhesion to FN/TNF-{alpha}, but also MMP-9 secretion, was of special interest in our study. Several recent studies have indicated that ligand binding and signaling via integrins may be important regulatory mechanisms of MMP expression and secretion [28 29 30 31 32 ]. It was shown that MMP-mediated T lymphocyte chemotaxis through an ECM gel is integrin-dependent because neutralizing antibodies to ß1, {alpha}4, and {alpha}5 integrin subunits block endogenous collagen-degrading activity [30 ]. Furthermore, Huhtala et al. [31 ] demonstrated that {alpha}5ß1 and {alpha}4ß1 act in a cooperative mechanism in which {alpha}4ß1 binding to the carboxy-terminal FN ligand disrupts the signal transduced through {alpha}5ß1, which induces MMP expression in fibroblasts. These findings strongly suggest that integrin-mediated adhesion and signaling are integral components in MMP gene regulation in various cell systems.

Our findings indicated that the {alpha}4ß1 and {alpha}5ß1 integrins, which mediate monocyte adhesion to FN, do not influence MMP-9 secretion. This raises the notion that the role of integrins in inducing MMP synthesis may be cell-, enzyme-, and context-specific. Macrophage interstitial collagenase (MMP-1) expression has been shown to be induced by insoluble collagen types I and III, whereas MMP-9 expression is unaffected by exposure to these substrates. In contrast, culturing macrophages on insoluble laminin and FN affected expression of neither of these enzymes [44 ], suggesting that integrin interactions with certain ECM moieties differentially influence which MMPs are expressed by macrophages and other cell types. This concept is also supported by the recent finding that a loss of cell adhesion and TNF-{alpha} stimulation synergistically increase collagenase expression in fibroblast-like synoviocytes [45 ]. Furthermore, there is evidence that integrin-mediated MMP synthesis may be differentially regulated when the FN molecule is in its native form versus its fragmented forms [32 ], as found in sites of inflammation [46 , 47 ]. Collectively, these results suggest that the changing context of the ECM environment during inflammation involves serial changes in structure and composition, thus yielding pleiotropic cellular responses, particularly cell adhesion and the secretion of MMPs. The FN/TNF-{alpha} complex may temporally represent a site for cell adhesion, as well as a mode for the ECM to localize potent induction of MMP-9 expression by immobilizing TNF-{alpha}.

Our previous studies on the T cell adhesion-strengthening effects of FN-bound TNF-{alpha} prompted us to investigate this same effect on monocytes. We postulated that the adhesion-stimulatory effects of FN-bound TNF-{alpha} could be the result of co-recognition of FN and TNF-{alpha} by their respective receptors, possibly leading to receptor crosstalk or the stabilization of adhesion through downstream intracellular signaling. However, in contrast to our previous findings with T cells [4 , 15 ], MonoMac-6 cell adhesion mediated by a primary stimulus, PMA, was not enhanced by FN-bound TNF-{alpha} (Fig. 5A) . These contrasting data suggest that, unlike in T lymphocytes, the signaling mechanisms underlying monocyte adhesion to FN/TNF-{alpha} are exclusively transmitted via integrins, not via crosstalk between integrins and TNF-{alpha} receptors. Moreover, TNF-{alpha} in its ECM-complexed form may serve in other functions of regulating monocyte behavior, such as the stimulated secretion of MMPs.

Several investigations have also highlighted the putative role of TNF-{alpha} in either promoting or preventing leukocyte migration. Although the cytokine is an inducer of monocyte transendothelial migration [48 ], it also has intriguing effects in leukocyte chemotaxis during inflammatory episodes through its combinatorial actions with those of other proinflammatory mediators. For example, it has been well-established that MCP-1 is a potent chemoattractant for monocytes, but TNF-{alpha} may regulate such movement by rapidly down-regulating the expression of CCR2, the receptor for MCP-1 [49 , 50 ]. This response of suppressing CCR2 receptor expression, divergent from the stimulatory effects of TNF-{alpha} on MCP-1 synthesis, may represent a mechanism for dually regulating chemokine activity. However, in the present study, we observed neither rapid (3 h) suppression of CCR2 expression by TNF-{alpha} nor significant reduction in expression (17 ± 2.5%) after prolonged (20 h) TNF-{alpha} treatment (data not shown). Although the reasons for these differences in CCR2 expression affected by TNF-{alpha} are unknown, it might be explained by the lower dose of TNF-{alpha} (1 ng/mL; 84 U/mL) used in our study compared to other studies [49 , 51 ]. Herein, we found that FN/TNF-{alpha} may cause rapid (3 h) retention of monocytes and prevent their chemotactic movement toward MCP-1 [49 ] through an unidentified mechanism. Although our findings indicated that chemotaxis through ECM gels toward MCP-1 was not arrested by complexed TNF-{alpha}, the longer duration of exposure to the cytokine (20 h), which results in significant MMP-9 secretion and accumulation (Fig. 3A) , may have led to a prominent role for MMPs in mediating migration. Moreover, although other studies have demonstrated a TNF-{alpha}-mediated decrease in migration across endothelium [50 ] or uncoated filters [49 ] toward MCP-1, our study suggests that the effects of TNF-{alpha} on chemotaxis may be context-dependent, since ECM gels with complexed TNF-{alpha} may serve as a substrate for migration and MMP activity. Thus, it seems plausible that short exposure to FN/TNF-{alpha} may transiently retain monocytes at inflammatory sites [49 , 51 ] to transmit TNF-{alpha}-mediated signals, such as MMP synthesis. Over time, the actions of FN/TNF-{alpha} on MMP-9 expression may cooperatively lead to a shift in MMP-mediated ECM degradation and migration toward MCP-1, as suggested by the greater inhibition of chemotaxis by GM6001 in the presence of bound TNF-{alpha} compared to MCP-1 alone (P < 0.05; not shown).

Considering the potent proinflammatory effects of TNF-{alpha}, its perpetuation at localized areas of inflammation may influence immune cell behavior in contrasting roles. Beneficially, ECM-bound TNF-{alpha} may stimulate and activate incoming immune cells, inducing effector functions. Such a system is especially conceivable in monocyte/macrophage physiology, which likely utilizes a regulatory autocrine loop of TNF-{alpha}-induced responses [20 ]. Recently, it was demonstrated that the prototypic proinflammatory cytokines TNF-{alpha} and IL-1ß selectively up-regulate expression of MMP-9 by monocytes and monocyte-derived macrophages, suggesting that these cells are actively involved in destruction of ECM [17 , 18 ]. Inducible expression of MMP-9 by FN/TNF-{alpha} may modulate monocyte movement through inflamed tissues, since MonoMac-6 cells express MMP-9 during migration toward MCP-1 [36 ]. Our study demonstrates that TNF-{alpha} secreted by monocytes is deposited into ECM after only 4 h, and that FN/TNF-{alpha} induces MMP-9 secretion up to 48 h after plating the cells. Thus, immediate release of TNF-{alpha}, followed by extended binding to ECM constituents may secure an injured tissue site with a potent stimulus of MMP secretion and possibly other mediators from immune cells, until inflammation is resolved. Such binding of TNF-{alpha} at the area of its secretion also immobilizes the cytokine, thereby limiting its availability and bioactivity proximal to an inflammatory locus. In contrast to its potential benefits, ECM-immobilized TNF-{alpha} may perpetuate inflammatory cell responses toward adverse conditions, causing dysregulated secretion of mediators such as MMPs. Indeed, TNF-{alpha}-induced MMP secretion is a primary cause of tissue destruction in various inflammatory diseases [45 , 52 53 54 ]. Further studies aimed at understanding the role of ECM in presenting a specific medley of signals, as provided by immobilized cytokines, to immune cells will likely contribute significant insight into acute and chronic inflammatory diseases.


   ACKNOWLEDGEMENTS
 
This work was supported by research grants from The Israel Science Foundation, founded by The Israel Academy of Sciences and Humanities, The Israel Cancer Research Fund (ICRF), and The Center for the Study of Emerging Diseases. G. G. Vaday is the recipient of a Feinberg Fellowship from the Weizmann Institute of Science. O. Lider is the incumbent of the Weizmann League Career Development Chair in Children’s Diseases.

Received April 22, 2000; revised June 17, 2000; accepted June 20, 2000.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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