Interferon- (cid:1) Differentially Regulates Monocyte Matrix Metalloproteinase-1 and -9 through Tumor Necrosis Factor- (cid:2) and Caspase 8*

Tumor necrosis factor- (cid:2) (TNF (cid:2) ) and granulocyte macrophage colony-stimulating factor (GM-CSF) individually enhance monocyte matrix metalloproteinase-9 (MMP-9) but induce MMP-1 only when added in combination. Because interferon- (cid:1) (IFN (cid:1) ) is also found at inflammatory sites, we determined its effect on monocyte MMPs in the presence or absence of TNF (cid:2) and GM-CSF. IFN (cid:1) alone did not stimulate monocyte MMP-9 or MMP-1; however, in the presence of GM-CSF it induced MMP-1 and enhanced MMP-1 stimulated by GM-CSF and TNF (cid:2) . IFN (cid:1) induced MMP-1 in the presence of GM-CSF through the stimulation of TNF (cid:2) production through a mechanism involving both p38 and ERK1/2 MAPKs, in which GM-CSF stimulated ERK1/2 whereas IFN (cid:1) activated p38. In support of this conclusion TNF (cid:2) neutralizing antibody and antibodies against TNF receptor I and -II blocked the induction of MMP-1 by GM-CSF and IFN (cid:1) . In contrast to its effects on MMP-1, IFN (cid:1) inhibited TNF (cid:2) -induced MMP-9 through a caspase tion suggested several mechanisms are potentially involved in the regulation of these enzymes. Previous studies (19, 20) have shown that IFN (cid:3) can influence TNF (cid:1) production by monocytes. IFN (cid:3) priming of monocytes enhances LPS- and bacillus Calmette-Guerin-induced production of TNF (cid:1) (19, 20). Addi-tionally, treatment of LPS-desensitized monocytes with IFN (cid:3) induces TNF (cid:1) production (21). Our findings demonstrate that combining IFN (cid:3) with GM-CSF results in a significant induction of TNF (cid:1) mRNA and protein. The increase in TNF (cid:1) in the presence of GM-CSF results in the induction of MMP-1 and also causes the enhancement of MMP-1 when IFN (cid:3) is added with TNF (cid:1) and GM-CSF. This conclusion is supported by the ability of neutralizing antibodies against TNF (cid:1) , TNFR-I, or TNFR-II to block the induction of MMP-1 by IFN (cid:3) and GM-CSF. Moreover, the stimulation of monocyte TNF (cid:1) by the combination of IFN (cid:3) and GM-CSF was shown to involve both the p38 and ERK1/2 MAPKs.

Monocytes/macrophages are prominent at sites of inflammation associated with extensive connective tissue destruction. They are thought to contribute to the loss of connective tissue components at these sites through the production of matrix metalloproteinases (MMPs). 1 MMPs are comprised of a family of extracellular matrix degrading enzymes that include the interstitial collagenases, gelatinases, stromelysins, matrilysin, metalloelastase, and membrane-type MMPs (1)(2)(3). MMP-1/interstitial collagenase and MMP-9/92-kDa gelatinase are major MMPs produced by monocytes. Fibrillar collagens, such as type I and II collagens, are degraded primarily by interstitial collagenases whereas MMP-9 is involved in degrading fibrillar collagens that have been denatured by the initial cleavage by MMP-1. Additionally, MMP-9 cleaves substrates, such as laminin and type IV collagen, that comprise the basement membrane.
As monocytes migrate into an inflammatory site they are exposed to multiple cytokines that influence the MMPs produced by these cells. The Th2 cytokines, IL-4 and IL-10, have been shown to suppress monocyte MMP production (4 -7). Inhibition of monocyte MMPs by these Th2 cytokines is related in large part to their suppression of prostaglandin H synthase-2 or cyclooxygenase-2 resulting in the loss of prostaglandin E 2 required for the induction of MMPs (4,6,8). A number of cytokines have also been shown to induce or enhance MMP production by monocytes. Depending on the specific cytokine or combination of cytokines, monocyte MMP production can be differentially regulated. For example, TNF␣, IL-1␤, or GM-CSF, when added individually to monocytes, enhance the expression of 92-kDa gelatinase (MMP-9) but not interstitial collagenase (MMP-1) (9,10). However, the combination of TNF␣ with GM-CSF induces the production of MMP-1 by monocytes through a prostaglandin-dependent pathway and synergistically enhances MMP-9 through a prostaglandin-independent mechanism (10).
Another cytokine produced by activated T cells and NK cells at inflammation sites is IFN␥ and has classically been considered an activator of monocytes. However, IFN␥ has been shown previously (9,11,12) to inhibit the production of MMPs by monocytes/macrophages stimulated with Con A or LPS. Because it is most likely that IFN␥ interacts in vivo with monocytes in the context of other cytokines, we hypothesized that its effect on monocyte MMPs may be different in the presence of other cytokines than with Con A or LPS. We therefore examined the effect of IFN␥ on the production of monocyte MMPs in the presence or absence of TNF␣ and/or GM-CSF, as well as the mechanisms through which IFN␥ might mediate its effects.
Here we report that IFN␥ in the presence of TNF␣ and/or GM-CSF differentially regulates the production of monocyte MMP-9 and MMP-1. Although IFN␥ alone does not stimulate monocyte MMP production, it induces MMP-1 when combined with GM-CSF and enhances the production of MMP-1 induced by GM-CSF and TNF␣. This occurs as a result of IFN␥-medi-ated increase in monocyte TNF␣ production when combined with GM-CSF and involves both p38 and ERK1/2 mitogenactivated protein kinases (MAPKs). In contrast, IFN␥ inhibits TNF␣-induced MMP-9 production through a novel mechanism involving caspase 8 regulation of STAT1 through p38 MAPK that is independent of apoptosis.

EXPERIMENTAL PROCEDURES
Purification of Human Monocytes-Human peripheral blood cells were obtained by leukapheresis of normal volunteers at the Department of Transfusion Medicine at the National Institutes of Health. These cells were diluted in endotoxin-free phosphate-buffered saline without Ca 2ϩ and Mg 2ϩ (BioWhittaker, Walkersville, MD) and layered over 20 ml of endotoxin-free lymphocyte sedimentation medium (Organon Teknika Corp., Durham, NC) in 50-ml tubes (BD Biosciences). After density sedimentation at 400 ϫ g for 30 min, the monocytes in the mononuclear cell layer were purified by counterflow centrifugal elutriation on a Beckman elutriation system as described previously (13,14), except that pyrogen-free phosphate-buffered saline was used in the elutriation procedure. Monocytes were enriched to Ͼ90% as determined by morphology, nonspecific esterase staining, and flow cytometry. Moreover, the purification procedure did not activate the monocytes as shown by the fact that following overnight incubation at 37°C in suspension less than 4% of these cells were IL-2 receptor-positive, a sensitive marker of monocyte activation (15).
Western Analysis of MMP-1 and MMP-9 -For detection of MMP-1 and MMP-9, the conditioned media from monocyte cultures (5 ϫ 10 6 /ml of Dulbecco's modified Eagle's medium) were harvested 36 to 48 h after the addition of cytokines and centrifuged. Bovine serum albumin (50 g/ml) was added to the culture supernatants prior to the precipitation of the proteins with cold ethanol (final concentration 60%) at Ϫ70°C for at least 15 min. Pelleted proteins (12,000 ϫ g for 20 min) were washed with 1 ml of ethanol and subsequently lyophilized by rotary evaporation. The lyophilized proteins were resuspended in SDS-Laemmli loading buffer (500 mM Tris-HCl (pH 6.8)/10% SDS/0.01% bromphenol blue/ 20% glycerol), reduced with 1% ␤-mercaptoethanol, heated for 4 min at 95°C, loaded, and electrophoresed on a 8 -16% Tris/glycine gradient polyacrylamide gel (Novex, San Diego, CA) in SDS running buffer (25 mM Tris-HCl (pH 8.3)/192 mM glycine/10% SDS). After electrophoresis, the proteins in the supernatants from conditioned media were transferred onto 0.45 m nitrocellulose in a buffer containing 25 mM Tris-HCl (pH 8.3)/192 mM glycine/20% methanol and blocked with TBST (50 mM Tris-HCl (pH 7.5)/150 mM NaCl/0.3% Tween 20) containing 5% nonfat dry milk for at least 1 h. The blots were washed three times with TBST and then incubated for 1 h or overnight with primary antibody. For the detection of MMP-1 and MMP-9, the blots were incubated with rabbit polyclonal antibodies against MMP-1 (generously provided by Dr. Henning Birkedal-Hansen, NIH, Bethesda, MD) and the N-terminal region of the active form of MMP-9 (Chemicon, Temecula, CA). Western blots were analyzed either by the addition of protein A-horseradish peroxidase (1:3000 dilution in TBST containing 5% nonfat dry milk; Amersham Biosciences) and developed with the ECL detection system (Amersham Biosciences) or by the addition of Alexa Fluor 680 goat anti-rabbit or anti-mouse (Molecular Probes Inc., Eugene, OR) and the infrared fluorescence detected with the Odyssey infrared imaging system (LI-COR, Lincoln, NE). The antibody against MMP-1 recognized the active and pro-collagenase forms.
Detection of TNF␣ and MMP-9 mRNA by Semi-quantitative RT-PCR-Total RNA was prepared from the culture cells using Trizol reagent according to the manufacturer's instructions (Invitrogen). The level of TNF␣ and MMP-9 mRNA was then determined using a semiquantitative one-step RT-PCR with GAPDH or ␤-actin as an internal control. RT-PCR was carried out using Promega's Access RT-PCR kit according to manufacturer's instructions (Promega, Madison, WI), and the PCR products were separated on a 1.4% agarose gel. Expression of TNF␣ and MMP-9 mRNA was normalized to that of GAPDH or ␤-actin mRNA, which was consistently expressed and not significantly affected by different cell culture conditions.
Detection of Soluble TNF␣ by Enzyme-linked Immunosorbent Assay-Purified human monocytes were treated with or without 50 ng/ml GM-CSF and/or IFN␥ for various times. The supernatants were analyzed for the production of TNF␣ by enzyme-linked immunosorbent assay according to the manufacturer's instructions (Immunotech, Marseille, France).

Differential Effect of IFN␥ on the Regulation of MMP-1 and MMP-9 Stimulated by TNF␣ and GM-CSF-
The association of IFN␥ with other cytokines, such as TNF␣ and GM-CSF, in chronic inflammatory lesions suggests that it may act in concert with these cytokines to modulate monocyte MMPs. Therefore, we tested the effect of IFN␥ on the production of monocyte MMPs in the presence or absence of TNF␣ and GM-CSF. IFN␥ alone failed to influence the production of MMP-1 and MMP-9 by monocytes (Fig. 1). However, synthesis of monocyte MMP-1, as detected by Western analysis, was induced when IFN␥ was combined with GM-CSF but not TNF␣ (Fig. 1). Moreover, the stimulation of monocyte MMP-1 by the combination of GM-CSF and TNF␣ was significantly enhanced by IFN␥ (Fig. 1). In contrast, as shown previously (9), IFN␥ suppressed the enhancement of MMP-9 by TNF␣. Although the lowest concentration of IFN␥ utilized in this experiment was 10 ng/ml, inhibition of TNF␣-induced MMP-9 was also observed with 0.5 to 10 ng/ml of IFN␥ (data not shown). However, the GM-CSFmediated increase in MMP-9 was further enhanced by IFN␥, whereas the 1.8-fold increase, as determine by densitometry, in MMP-9 stimulated by the combination of TNF␣ and GM-CSF was slightly decreased by IFN␥. These results demonstrate that whereas IFN␥ alone has no effect on monocyte MMP-1, it can significantly effect the induction or enhancement of MMP-1 when combined with other cytokines, whereas IFN␥ can have stimulatory or inhibitory effects on MMP-9 depending on the specific cytokines present.
Effect of Time of Addition of IFN␥ on MMP-1 and MMP-9 Induction-In light of the ability of IFN␥ to induce or enhance MMP-1 when added with GM-CSF or GM-CSF plus TNF␣, respectively, IFN␥ was added at various times with respect to the other cytokines to determine the time course of the IFN␥ modulatory effects. As shown in Fig. 2A, when IFN␥ was added at the indicated times with GM-CSF the optimal induction occurred if these two cytokines were added at the same time. When IFN␥ was added 1 h prior to GM-CSF it was not as effective at inducing MMP-1, and when added 4.5 h after GM-CSF the effect of IFN␥ was essentially lost. Similarly, when IFN␥ was added 1 h before the combination of GM-CSF and TNF␣ it further enhanced MMP-1 production, but the optimal response, as with IFN␥ and GM-CSF, occurred when all three cytokines were added at the same time. As with GM-CSF, the effect of IFN␥ was lost when it was added 4.5 h after the combination of GM-CSF and TNF␣. Examination of the timedependent effects of IFN␥ on GM-CSF-induced MMP-9 revealed a kinetic pattern similar to that observed with MMP-1 (Fig. 2B). In contrast, when added with GM-CSF and TNF␣, IFN␥, in general, suppressed MMP-9 production. Because the suppressive effects of IFN␥ on GM-CSF and TNF␣-induced MMP-9 were not lost by 4.5 h, and may be related to its suppression of TNF␣-stimulated MMP-9 as shown in Fig. 1, we examined the effect of IFN␥ on TNF␣-induced MMP-9 at later time points. As shown in Fig. 2C, IFN␥ suppressed TNF␣induced MMP-9 when added as late as 4 to 8 h after TNF␣. The failure of IFN␥ to inhibit MMP-9 after 8 h was related to the induction of MMP-9 mRNA above control (media only) levels by TNF␣ between 8 to 12 h (data not shown).
IFN␥ and GM-CSF Stimulate Monocyte MMP-1 and MMP-9 through the Induction of TNF␣-A potential mechanism by which IFN␥ may stimulate MMP-1 in the presence of GM-CSF and enhance MMP-9 may be because of induction of another cytokine(s). We tested this possibility through the addition of neutralizing antibodies. As shown in Fig. 3A, neutralizing an-tibodies against TNF␣ blocked the induction of MMP-1 and the enhancement of MMP-9 by IFN␥ and GM-CSF. Moreover, antibodies against TNFR-I and -II also significantly decreased MMP-1 and MMP-9 production indicating that TNF␣ utilized both receptors in the stimulation of monocyte MMPs. These findings suggested that IFN␥, when added with GM-CSF, stimulated TNF␣ production that, in turn, resulted in the induction of MMP-1 and enhancement of MMP-9. As shown in Fig. 4, A  and B, whereas GM-CSF or IFN␥ caused a slight increase in TNF␣ mRNA and protein, when these cytokines were added in combination to monocytes there was an 11-fold increase in  TNF␣ mRNA and protein. Stimulation of TNF␣ production was detected 7 h after the addition of GM-CSF and IFN␥ with a substantial increase in TNF␣ occurring between 17 and 28 h (Fig. 5A). Addition of IFN␥ at varying times relative to GM-CSF revealed that when IFN␥ was added 4.5 h after GM-CSF its ability to induce TNF␣ was reduced by ϳ67% (Fig. 5B). These findings demonstrate that the effect of IFN␥ on monocyte MMP production when combined with GM-CSF is mediated through the stimulation of TNF␣ and explain why this effect is lost after 4.5 h.
GM-CSF and IFN␥ Induce TNF␣ through p38 and ERK1/2 MAPKs-To determine the possible mechanism(s) by which GM-CSF and IFN␥ induce TNF␣, we examined the role of MAPKs in the regulation of TNF␣. Addition of the p38 inhibitor, SB203580, or the ERK1/2 inhibitor, PD98059, suppressed the level of TNF␣ detected in the media (Fig. 6A). PD98059 caused the greatest inhibition of TNF␣. Examination of the activation of p38 and ERK1/2 revealed that GM-CSF increased ERK1/2 phosphorylation whereas IFN␥ enhanced the activation of p38 (Fig. 6B). These results demonstrate that both p38 and ERK1/2 are required for TNF␣ production by IFN␥ and GM-CSF. Fig. 1, unlike its ability to increase MMP-1 and MMP-9 in the presence of GM-CSF, IFN␥ suppressed TNF␣-induced MMP-9 production. Because recent studies have shown that IFN␥ can activate caspase 8 in other cell types (16,17), we examined whether activation of caspases might be the mechanism through which IFN␥ suppresses TNF␣-mediated stimulation of monocyte MMP-9. Addition of an inhibitor of caspases 6, 8, 9, and 10 to TNF␣-and IFN␥-treated monocytes resulted in a significant restoration of MMP-9 as shown by Western (Fig. 7A) and zymogram analysis (Fig. 7B). An inhibitor of caspases 3, 6, 7, 8, and 10 also restored MMP-9 production, although to a lesser degree, because this is primarily a caspase 3-inhibitor (Fig. 7A). In contrast, an inhibitor of caspases 1 (interleukin 1␤-converting enzyme) and 4 failed to restore MMP-9 production (Fig. 7A).

Caspase 8 Inhibitors Reverse IFN␥-mediated Inhibition of TNF␣-induced MMP-9 -As shown in
We further examined the individual inhibitors against caspase 6, 8, and 9, as well as a general caspase inhibitor. The inhibitor of caspase 8 activity, as well as a general inhibitor (all), but not caspase 9, reversed the inhibition by IFN␥-of TNF␣-induced MMP-9 production (Fig. 7B). This was shown to be because of inhibition at the transcriptional level as demonstrated by restoration of MMP-9 mRNA by an inhibitor of caspase 8 (Fig. 7C).
Stimulation and Activation of Caspase 8 by IFN␥-The caspase inhibitor studies indicated that IFN␥ was mediating its inhibitory effect on TNF␣-stimulated MMP-9 through a caspase pathway. To examine this possibility we next determined the effects of IFN␥ on the activation of caspase 8, because it is an apical caspase involved in downstream effects on other caspases, and it was shown by our inhibitor studies to be involved in restoration of MMP-9. Western analysis revealed that IFN␥ or TNF␣ increased the 55-kDa proenzyme form of caspase 8, as well as the first cleavage products, which are 45/43-kDa fragments (Fig. 8A). However, only in the presence of IFN␥ were significant amounts of the p25-kDa fragment detected, which indicates an increase of the active p18 fragment of caspase 8 that was not detected with this antibody, as both p25-and p18-kDa fragments are derived from cleavage of the p43-kDa fragment. However, the p18 fragment was detected with a specific antibody against this fragment and was only induced in the presence of IFN␥ (Fig. 8B). In contrast, stimulation with TNF␣ alone failed to result in substantial amounts of the p25 or p18 fragment of caspase 8.

Caspase 8 Inhibition Suppresses IFN␥-induced STAT1
Phosphorylation-IFN␥ mediates many of its effects on cellular function through the phosphorylation of STAT1. Moreover, recently IFN␥-induced phosphorylation of STAT1 has been shown to be involved in the inhibition of TNF␣-stimulated MMP-9 in human Ewing's sarcoma EW-7 cells (18). We there- fore examined the effect of an inhibitor of caspase 8 on IFN␥stimulated phosphorylation of STAT1 in monocytes. As shown in Fig. 9, IFN␥ treatment of monocytes in the presence or absence of TNF␣ induced the phosphorylation of STAT1. Addition of a caspase 8 inhibitor suppressed the IFN␥-mediated phosphorylation of STAT1. These findings indicate caspase 8 is involved in the regulation of the phosphorylation of STAT1.
IFN␥-activated Caspase 8 Regulates Phosphorylation of STAT1 through p38 MAPK-To determine the mechanism through which IFN␥ activation of caspase 8 regulates STAT1 phosphorylation we initially examined the potential role of p38 and ERK1/2 MAPKs in IFN␥-mediated phosphorylation of STAT1. As shown in Fig. 10A, IFN␥ stimulated the phosphorylation of p38, as also shown in Fig. 6B; however, IFN␥ did not increase the phosphorylation of ERK1/2. Involvement of p38 in the phosphorylation of STAT1 was demonstrated through the use of SB203580, a specific inhibitor of p38 MAPK, which suppressed the phosphorylation of STAT1 by IFN␥ (Fig. 10B). In contrast, PD98059, an inhibitor of ERK1/2, had no affect on the phosphorylation of STAT1.
Caspase 8 Regulates the Activation of p38 by IFN␥ and TNF␣-Because IFN␥ induction of p38 activity was involved in the phosphorylation of STAT1 we next examined the effect of caspase 8 inhibition on the phosphorylation of p38 by IFN␥ and the combination of TNF␣ and IFN␥. As shown in Fig. 11A, IFN␥-induced phosphorylation of p38 was suppressed by inhibition of caspase 8. In contrast, inhibitors of caspase 6 and 9 had little affect on the phosphorylation of p38 by IFN␥ (Fig.  11A). Additionally, the phosphorylation of p38 by TNF␣ plus IFN␥ was suppressed by an inhibitor of caspase 8 and a general caspase inhibitor (Fig. 11B).
Monocyte Apoptosis Is Unaffected by IFN␥-Activation of caspase 8 is generally associated with apoptosis that could account for the suppression of MMP-9 stimulated by TNF␣. However, analysis of annexin V, a marker of early apoptosis, demonstrated that IFN␥ did not cause apoptosis of monocytes (Fig. 12). Although the level of apoptosis in control cells in this representative experiment was 17%, the levels of apoptotic cells in cultures treated with IFN␥ and/or TNF␣ ranged from 6 to 13%. These results indicate that stimulation of monocytes with either IFN␥ or TNF␣ does not increase apoptosis as com-FIG. 6. Induction of TNF␣ by GM-CSF and IFN␥ occurs through p38 and ERK1/2 MAPKs. A, monocytes were plated at 5 ϫ 10 6 /ml in 12-well plates for 30 min and then incubated in the presence or absence of SB203580 or PD98059 for 30 min prior to the addition of 50 ng/ml GM-CSF and IFN␥. Culture supernatants were harvested 24 h after the addition of cytokines and assayed for TNF␣ by enzyme-linked immunosorbent assay. B, monocytes were plated at 20 ϫ 10 6 /4 ml in 60-mm Petri dishes for 30 min prior to the addition of 50 ng/ml of IFN␥ and/or GM-CSF. Cell lysates acquired 1 h after cytokine stimulation were fractionated by SDS-PAGE on 12% Tris/glycine gels and assayed for phosphorylated and total p38 and ERK1/2 by Western blot. pared with control cells. Moreover, the number of dead cells, as detected by propidium iodide, was not increased by either IFN␥ or TNF␣ (data not shown). These findings demonstrate that the inhibitory effect of IFN␥ on TNF␣-stimulated MMP-9 is not because of apoptosis of the monocytes.

DISCUSSION
The findings presented here demonstrate that although IFN␥ alone does not affect the production of MMP-1 or MMP-9 by monocytes; however, in the presence of GM-CSF or TNF␣ it differentially regulations the production of these MMPs. IFN␥ in combination with GM-CSF induces the production of MMP-1. This occurs as a result of the stimulation of TNF␣ synthesis by the combination of GM-CSF and IFN␥ that, in turn, acts with GM-CSF to stimulate MMP-1 production. In contrast to MMP-1, IFN␥ inhibits the stimulation of MMP-9 by TNF␣ through a caspase 8/p38/STAT1 pathway.
The differential effects of IFN␥ on monocyte MMP produc- Purified monocytes were treated with IFN␥ (50 ng/ml) and/or TNF␣ (50 ng/ml). A, following stimulation of monocytes for 3 h with cytokines, 100 g of cytosolic protein was analyzed for cleavage fragments of caspase 8 by Western blot analysis with an antibody that detects 55/53-kDa proenzyme form of caspase 8, the 45/43-kDa first cleavage product, and the p25-kDa form. B, the active p18 fragment generated from p25 was detected with an antibody specific for this fragment. tion suggested several mechanisms are potentially involved in the regulation of these enzymes. Previous studies (19,20) have shown that IFN␥ can influence TNF␣ production by monocytes. IFN␥ priming of monocytes enhances LPS-and bacillus Calmette-Guerin-induced production of TNF␣ (19,20). Additionally, treatment of LPS-desensitized monocytes with IFN␥ induces TNF␣ production (21). Our findings demonstrate that combining IFN␥ with GM-CSF results in a significant induction of TNF␣ mRNA and protein. The increase in TNF␣ in the presence of GM-CSF results in the induction of MMP-1 and also causes the enhancement of MMP-1 when IFN␥ is added with TNF␣ and GM-CSF. This conclusion is supported by the ability of neutralizing antibodies against TNF␣, TNFR-I, or TNFR-II to block the induction of MMP-1 by IFN␥ and GM-CSF. Moreover, the stimulation of monocyte TNF␣ by the combination of IFN␥ and GM-CSF was shown to involve both the p38 and ERK1/2 MAPKs.
Unlike MMP-1 in which TNF␣ fails to induce this enzyme in monocytes, TNF␣ alone induces MMP-9. However, as shown here and previously (9), IFN␥ suppresses the induction of MMP-9 by TNF␣. In contrast, when IFN␥ was added with GM-CSF it enhanced MMP-9 production through the induction of TNF␣. This may result from the failure to totally inhibit the additive effect of GM-CSF with the endogenously produced TNF␣ that is significantly enhanced beginning at 7 h after the addition of IFN␥. Additionally, as opposed to experiments in which IFN␥ inhibited MMP-9 when added at the same time as TNF␣, the GM-CSF-and IFN␥-mediated increase of TNF␣ occurred several hours after the major suppressive effects of IFN␥ on the induction of MMP-9. Thus, production of TNF␣ induced from 7 to 28 h after IFN␥ stimulation resulted in further enhancement of GM-CSF-stimulated MMP-9 as measured at 48 h.
To determine the mechanism by which IFN␥ inhibited the induction of MMP-9 by TNF␣ our studies focused on caspases. Recent studies (16,17) have shown that IFN␥ can increase caspase 8 activity in Fanconi anemia group C hematopoietic progenitor cells and breast tumor cells, which results in apoptosis of these cells. Caspase 8 is an apical caspase that acti-vates caspase 3 and caspase 7 with caspase 3, in turn activating caspase 6 and caspase 2. These activated caspases cleave structural elements of the cytoplasm and nucleus and certain protein kinases that, in general, result in the disruption of survival pathways leading to apoptotic cell death (22,23). Our findings demonstrate that IFN␥ also activates caspase 8 in monocytes. However, IFN␥ activation of caspase 8 in monocytes does not lead to apoptosis. Of particular interest is the finding that inhibitors of caspase 8 reversed the inhibition of MMP-9 by IFN␥ in TNF␣-activated monocytes.
The proposed mechanism by which IFN␥ inhibits MMP-9 through a caspase 8 pathway is outlined in Fig. 13. We show that caspase 8 activated by IFN␥ increases the phosphorylation of p38 MAPK that, in turn, leads to the serine phosphorylation of STAT1. Evidence for this part of the pathway is supported by the ability of inhibitors of caspase 8 to block IFN␥-mediated phosphorylation of p38 MAPK and STAT1 and the suppression of STAT1 phosphorylation by the p38 inhibitor SB203580. Regulation of p38 MAPK by caspase 8 has been demonstrated previously (24) in a caspase 8-deficient cell line. Additionally, the activation of MEKK-1, the upstream activator of the Jun N-terminal kinases, by caspases provides further evidence for the involvement of caspases in the regulation of MAPKs (25,26). IFN␥-mediated phosphorylation of STAT1 has been shown to require p38 MAPK (27) but has also been reported to occur through a different signaling pathway (28). Our studies agree with the requirement of p38 for IFN␥-mediated phosphorylation of STAT1. The differences between these studies may be because of variations in the signaling pathways in the cell types used.
The proposed mechanism by which phosphorylated STAT1 inhibits TNF␣ induction of MMP-9 is based on data from previous studies. It has been shown in TNF␣-treated human Ewing's sarcoma EW-7 cells, transfected with the MMP-9 promoter, that phosphorylation of STAT1 by IFN␥ can inhibit MMP-9 production through the induction of interferon regulatory factor-1 (IRF-1); IRF-1 suppresses TNF␣-induced MMP-9 expression by binding to IFN␥-stimulated responsive elements that overlap NF-B binding sites on the MMP-9 promoter (18). Thus IRF-1 inhibits MMP-9 by completing with TNF␣-induced NF-B binding to the MMP-9 promoter. Further support for the suppressive role of STAT-1 on MMP-9 production comes from experiments in which reconstitution of STAT1 in STAT1-defi- FIG. 13. Proposed mechanism by which IFN␥ inhibits the induction of MMP-9 by TNF␣. The data presented here demonstrate that IFN␥ activation of caspase 8 leads to the phosphorylation of p38 that, in turn, is involved in the phosphorylation of STAT1. Based on previous findings, STAT1-induced IRF-1 has been shown to bind to the interferon-␥ stimulated responsive elements that overlap with the NF-B binding site in MMP-9 thus inhibiting the TNF␣-induced MMP-9 production. cient tumor cells decreased expression of MMP-9 (29). Our findings are in agreement with these studies and provide an additional signaling component, caspase 8, as a regulator of STAT1 phosphorylation leading to the IFN␥-induced suppression of MMP-9. These data demonstrate a new and novel effect of IFN␥ on the regulation of MMPs, at least in part, through a caspase 8 pathway that is independent of apoptosis.
In summary, the findings presented here demonstrate that the effect of IFN␥ on the regulation of monocyte involvement in connective tissue turnover is complex and is dependent on the cytokines present at an inflammatory site. Of significance is (a) the ability of IFN␥, in combination with GM-CSF, to stimulate the production of TNF␣ causing the induction of MMP-1 and (b) the activation of caspase 8 by IFN␥ resulting in the stimulation of a p38-STAT1 pathway that inhibits TNF␣-stimulated MMP-9 production. The mechanism by which caspase 8 activates p38 is currently under investigation.