Regulation of Hepatic Fibrosis and Extracellular Matrix Genes by the Th Response: New Insight into the Role of Tissue Inhibitors of Matrix Metalloproteinases

Brian Vaillant, Monica G. Chiaramonte, Allen W. Cheever, Paul D. Soloway and Thomas A. Wynn
J Immunol December 15, 2001, 167 (12) 7017-7026; DOI: https://doi.org/10.4049/jimmunol.167.12.7017

Abstract

Hepatic fibrosis is the hallmark of Schistosoma mansoni infection and often results in portal hypertension and bleeding from esophageal varices. The fibrotic process is highly dependent on type 2 cytokines, yet their role in the regulation of extracellular matrix remodeling genes remains largely unknown. Here, we examined the expression of matrix metalloproteases (MMP) -2, -3, -9, -12, and -13 and their inhibitors, tissue inhibitor of metalloproteases (TIMP) -1, -2, and -3, in the livers of infected mice and correlated their expression profiles with fibrosis and type 2 cytokine production. Expression of MMP-2, -3, -9, -12, and -13 and of TIMP-1 and -2 mRNA rapidly increased at the onset of egg laying in infected mice, while TIMP-3 was unchanged. Because TIMP are presumed to be important regulators of the extracellular matrix, and their expression correlated with the development of fibrosis, we studied their role in fibrogenesis by infecting TIMP-1- and TIMP-2-deficient mice. Strikingly, our data revealed no role for TIMP-1 or -2 in the fibrotic pathology induced by S. mansoni eggs. Because of these findings, we infected IL-10/IFN-γ-deficient mice that develop an exaggerated fibrotic response to determine whether changes in type 2 cytokine dominance influence the pattern of MMP and TIMP expression. Fibrosis and type 2 cytokine production correlated with increased MMP-2/MMP-9 vs TIMP-1/TIMP-2 expression. These data, in addition to our knockout studies, demonstrate that TIMP-1/TIMP-2 play no essential role in fibrogenesis in schistosomiasis. Indeed, our findings suggest that inhibiting profibrotic cytokines or specific MMP may be a more effective strategy to ameliorate fibrotic pathology.

Fibrosis is the process of excessive deposition of collagen and other extracellular matrix (ECM)3 components. Some ECM deposition is necessary for wound healing to provide strength and temporary structure to damaged tissues; however, if not limited, it can be pathologic. Liver fibrosis can be particularly detrimental, leading to portal hypertension and its attendant sequelae, which include splenomegaly and rupture-prone gastroesophageal varices. One fibrotic hepatic disease, schistosomiasis, is caused by the helminthic parasite Schistosoma mansoni and other related species. Interestingly, the healthy adult worm is only weakly immunogenic; the principal immune response is directed against the eggs, which the parasite produces in large numbers. Some of these eggs enter the portal circulation and become lodged in hepatic portal venules, where they initiate an immune response that often leads to fibrosis.

In C57BL/6 mice, the hepatic granulomatous response to S. mansoni eggs begins as a Th1-type response that is rapidly driven by egg Ag to a Th2-dominant response (1, 2, 3, 4). This Th2 response induces circumoval granulomas, which are rich in eosinophils and collagen fibers. IL-4 and IL-13 have been shown to be essential cytokines that promote fibrosis in this disease (5, 6). Altering the usual Th2 response by cytokine manipulation is informative. IL-10/IL-4 double-deficient mice develop a sustained Th1 response during infection. Granulomas from these mice are similar to normal C57BL/6 granulomas in size, but, in contrast, are almost entirely lacking in both eosinophils and fibrosis at 8 wk postinfection (7). This suggests that the Th2 response is profibrogenic in the schistosomiasis model. Furthermore, infected IL-10-deficient mice show a sustained mixed Th1/2 response, with elevated Th1 and Th2 cytokines, vs infected WT mice (7). This study and others suggest that IL-10 not only suppresses the Th1 response, but also plays a role in mitigating the Th2 response.

Because Schistosoma species are a significant worldwide cause of morbidity and mortality, there is considerable impetus to better understand the pathogenesis of the disease. Certain aspects of murine schistosomiasis are well characterized, such as the Th response to the eggs and the roles of specific cytokine mediators; however, one part of the disease process has been poorly studied, namely the proximal mediators of the fibrotic response, which act downstream of profibrogenic cytokines. Based on current understanding of ECM regulation, these mediators include matrix metalloproteases (MMP) and their specific inhibitors, tissue inhibitors of metalloprotease (TIMP).

MMP are a family of zinc- and calcium-dependent proteases that in concert degrade virtually all components of the ECM. They can be grouped into various categories based on substrate preference, shared structural motifs, or sequence homology. One broad grouping, based on substrate specificity, generates four categories: collagenases that degrade the major structural collagens, stromelysins that digest a wide array of ECM substrates, gelatinases capable of degrading denatured collagens and many components of basement membranes, and membrane-type metalloproteases anchored in the plasma membrane that can digest structural collagens and proteolytically activate other MMP (8, 9, 10, 11). MMP-12, which typically is not classified into the previous groups, degrades elastin and some basement membrane components (12, 13). MMP activity is primarily regulated at three levels: transcriptional control, proteolytic cleavage of the pro form to the active form, and inhibition by physiologic protease inhibitors, such as α2-macroglobulin and TIMP (14).

In both man and mouse there are currently four described TIMP, with expression of at least one in most adult tissues (15). The various TIMP are thought to inhibit all activated MMP to varying degrees, although few studies have systematically studied the inhibition of every MMP by every TIMP (16, 17, 18); however, some unique TIMP/MMP interactions have been characterized. TIMP-1 can form a complex with pro-MMP-9, and both TIMP-2 and -4 can bind to the pro form of MMP-2 (19, 20, 21). TIMP-2 can also form a trimolecular cell surface complex with pro-MMP-2 and MT1-MMP, which is important in activating pro-MMP-2 (22). TIMP-1 and -2 also promote the growth of several cell types, including erythroblasts, keratinocytes, and fibroblasts; this ability was shown to be independent of TIMP metalloprotease inhibition (23, 24, 25, 26). Anti-angiogenic roles for TIMP-1, -2, and -3 have also been described (27, 28, 29, 30). Finally, additional studies have suggested that TIMP-1 and -2 are anti-apoptotic for some cell types, whereas TIMP-3 may be pro-apoptotic (28, 31, 32, 33). Regarding fibrosis, the conventional wisdom is that TIMP, via their MMP inhibitory activity, are important for the accumulation of ECM. Thus, one would predict that in a fibrotic disease such as schistosomiasis, expression of TIMP would be elevated, whereas expression of MMP would be decreased.

To better understand the role of TIMP in the fibrotic pathology of schistosomiasis, we first examined their expression profiles in the liver following infection. We found that the expression of TIMP-1 and -2 correlated with the fibrotic response. We then directly addressed the relative importance of TIMP-1 and TIMP-2 by infecting knockout animals. We also examined several MMP to determine how their expression patterns changed during the course of infection. Finally, we infected mice that developed an exaggerated Th2 response to see how changes in fibrosis and type 2 cytokine production correlated with MMP and TIMP expression in vivo. Unexpectedly, our data revealed no significant role for TIMP-1 and TIMP-2 in the pathogenesis of schistosomiasis and instead pointed to MMP-2 and MMP-9 as potential downstream mediators of the disease.

Materials and Methods

Mice and infections

C57BL/6, IFN-γ-deficient mice, and IL-10-deficient mice were obtained from Taconic Farms (Germantown, NY). IL-10/IFN-γ double-deficient mice were generated by crossing IL-10 and IFN-γ knockout mice. TIMP-1-deficient (N9) and TIMP-2-deficient (N6) breeding pairs were backcrossed onto the C57BL/6 background (34, 35). Mice were infected percutaneously with ∼25 S. mansoni cercaria of the NMRI strain, acquired from Biomedical Research Institute (Rockville, MD). All mice were housed and handled according to approved National Institutes of Health animal protocols.

Histologic analysis

Determination of the collagen content of the liver by hydroxyproline measurement was described previously (5). Approximately half the liver was fixed in Bouin-Hollande solution, and histologic sections were processed and stained with Giemsa (Histo-Path of America, Clinton, MD). The diameters and eosinophil contents of granulomas (30 per mouse) surrounding single, mature, viable eggs were measured using an ocular micrometer, and the volume of each granuloma was calculated assuming a spherical shape. Eggs in the liver and intestines were counted separately after digestion in 4% KOH at 37°C. Other histologic parameters, such as the abundance of eosinophils (expressed as a percentage of total granuloma cells) and mast cells (expressed on a scale of 1–4), were determined by a pathologist.

Cell culture

Cells were extracted by homogenizing mesenteric lymph node (MLN) from infected mice and passage through a 100-μm pore size filter. Cells were washed; resuspended in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 50 μM 2-ME, and antibiotic-antimycotic solution (all from Life Technologies, Gaithersburg, MD); and plated into 24-well culture plates at 3 × 106 cells/well. Cells were placed in medium alone or stimulated with one of the following: soluble S. mansoni egg Ag (SEA; 20 μg/ml), soluble worm Ag preparation (SWAP; 40 μg/ml), or Con A (1 μg/ml) (5). After a 72-h incubation at 37°C, cell supernatants were harvested and frozen at −20°C until use.

ELISA for cytokines

Plates (96-well) were coated overnight with appropriate capture Ab against murine IFN-γ, IL-5, or IL-10 (all from BD PharMingen, San Diego, CA). After washing with 1× PBS and 0.05% Tween 20 and blocking plates for 2 h with 5% nonfat dry milk in 1× PBS and 0.05% Tween 20, supernatants were placed in wells and left overnight at 4°C. Plates were then washed and treated with the appropriate anti-cytokine Ab (rabbit anti-murine IFN-γ, biotinylated anti-murine IL-5, or biotinylated anti-murine IL-10 (all from BD PharMingen)) for 1 h, washed, and incubated with HRP-coupled donkey anti-rabbit Ig Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) for IFN-γ or with HRP-streptavidin conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for IL-10 or IL-5. After a final wash, substrate was added and developed for 15–30 min. Plate absorbance was read at 543 nm on a SpectraMax 190 (Molecular Devices, Sunnyvale, CA). IL-13 ELISA kits were purchased from R&D Systems (Minneapolis, MN). Supernatants and plates were processed according to the manufacturer’s protocol. IL-4 was assayed in supernatants as previously described, using CT4S cell incorporation of tritiated thymidine for quantitation (5).

RT-PCR

Mouse liver samples that had been stored at −70°C in 1 ml RNA STAT60 (Tel-Test, Friendswood, TX) were thawed, and RNA was prepared according to the manufacturer’s instructions. cDNA was prepared as previously described (36). PCR was performed in a Lightcycler thermocycler using the DNA master SYBR Green I Amplification Kit (both from Roche Molecular Biochemicals, Basel, Switzerland) and the appropriate primer set for the gene of interest (Table I⇓) for semiquantitative comparisons of gene expression. All samples were normalized to hypoxanthine phosphoribosyltransferase (HPRT), and data are presented in arbitrary units.

View this table:
Table I.

RT-PCR primers used in this study

Statistical analysis

For data analysis, the GraphPad PRISM program (GraphPad, San Diego, CA) was used to analyze groups using the Mann-Whitney test. Groups with p < 0.05 were considered significant. All experiments were repeated at least once with similar results.

Results

TIMP-1 and -2 mRNA expression increases in the livers of S. mansoni-infected mice

C57BL/6 mice were infected with S. mansoni, and liver tissue was harvested at successive time points to determine how TIMP mRNA expression is regulated throughout the acute and chronic stages of infection. mRNA expression in individual mice was assessed by real-time RT-PCR. Message for TIMP-1 was almost undetectable in uninfected mouse liver (Fig. 1⇓A). A small, but significant, increase in TIMP-1 mRNA was observed at the onset of egg laying (wk 6). Expression continued to rise through wk 12 and remained elevated, but at reduced levels, by wk 16. TIMP-2 was detected at low levels in uninfected mice (Fig. 1⇓B), but showed slightly different kinetics than TIMP-1. TIMP-2 expression peaked at wk 9 and then decreased by wk 12. By wk 16, TIMP-2 had returned to baseline levels despite ongoing schistosome infection and increasing levels of liver fibrosis (see hatched line in Fig. 1⇓A). In marked contrast, TIMP-3 mRNA was easily detectable in uninfected livers and showed little change following infection (Fig. 1⇓C). Finally, TIMP-4 mRNA was almost undetectable in both uninfected and infected mouse liver, but was, however, readily amplified from normal brain tissue (data not shown). Thus, TIMP-1 and -2 were the only TIMP that showed a high degree of regulation following infection.

FIGURE 1.
FIGURE 1.

Kinetics of TIMP expression in infected C57BL/6 mice. Time course of liver mRNA expression for TIMP-1 (A), TIMP-2 (B), and TIMP-3 (C) in S. mansoni-infected C57BL/6 mice. Expression values for each mouse have been normalized to the corresponding HPRT value. UI, uninfected mice. ∗, p < 0.05 vs the uninfected group. Each data point represents one mouse, while bars represent the median value for each group. The line in A denotes a representative pattern of liver fibrosis during the same time frame (graphed as the fold change in hydroxyproline levels per worm pair).

Absence of a role for TIMP-1 or -2 in the pathogenesis of schistosomiasis

Many studies have postulated an important role for TIMP in the regulation of the ECM during chronic inflammatory reactions (37, 38, 39). Therefore, to more directly examine the role of TIMP in the regulation of schistosome egg-induced pathology, we infected TIMP-1- and -2-deficient mice. Animals were sacrificed at both the acute and chronic stages postinfection to assess granuloma formation and hepatic fibrosis.

For RT-PCR studies, animals were infected for 9 wk, and gene expression was examined in the livers of individual mice by real-time PCR. Infected C57BL/6 and TIMP-2-deficient mice showed an up-regulation in TIMP-1 mRNA levels over uninfected animals (Fig. 2⇓A, upper panel). Of interest, TIMP-2-deficient mice displayed significantly reduced levels of TIMP-1 mRNA vs the infected WT controls; thus, these animals were manifesting what could be considered a dual deficiency in TIMP-1 and -2. The wild-type (WT) C57BL/6 and TIMP-1-deficient mice also displayed elevated TIMP-2 mRNA following infection (Fig. 2⇓A, lower panel); however, in contrast to the TIMP-2-deficient mice, there was no evidence that TIMP-2 mRNA was affected by TIMP-1 deficiency. Again, consistent with previous experiments, there was no change in TIMP-3 expression in any group (data not shown). All MMP were expressed constitutively at low levels before infection, but were significantly induced following schistosome challenge (Fig. 2⇓B). No significant differences were detected between the WT and TIMP-1-deficient animals; however, MMP-2, -3, -12, and -13 were all significantly decreased in the TIMP-2-deficient mice. No significant differences were observed for MMP-9, although all three groups showed a marked up-regulation following infection.

FIGURE 2.
FIGURE 2.

Expression of TIMP in TIMP-1-deficient and TIMP-2-deficient mice. Liver mRNA expression for TIMP-1 and TIMP-2 (A) or MMP (B) in mice infected with S. mansoni for 9 wk in C57BL/6 (□), TIMP-1 knockout (▵), and TIMP-2 knockout (▿) mice. Filled shapes represent infected mice; open shapes represent uninfected mice (UI). Expression values for each mouse have been normalized to the corresponding HPRT value. Mann-Whitney test p values vs C57BL/6 are shown. Each data point represents one mouse, while bars represent the median value for each group.

Finally, the livers of infected TIMP-1 and -2-deficient mice were examined at 9 and 14 wk postinfection to determine whether granuloma formation or fibrosis was affected by TIMP deficiencies. WT and TIMP-1-deficient mice showed a similar degree of hepatic fibrosis, while fibrosis increased slightly, but significantly, in the TIMP-2-deficient mice on wk 9 (Fig. 3⇓A). Nevertheless, by wk 14 all three groups were indistinguishable in terms of liver collagen content (Fig. 3⇓B). There was also no evidence for a significantly altered inflammatory response in the TIMP-1 or -2-deficient mice, because granuloma volumes did not deviate from WT at 9 (Fig. 3⇓C) or 14 wk postinfection (Fig. 3⇓D). It is notable, however, that the number of granuloma-associated mast cells decreased significantly in the TIMP-2-deficient mice at both 9 wk (C57BL/6, 2.9 ± 0.386 (n = 10); TIMP-2-deficient, 1.9 ± 0.386 (n = 10)) and 14 wk (C57BL/6, 2.8 ± 0.310 (n = 10); TIMP-2-deficient, 1.75 ± 0.293 (n = 8)). These data illustrate that neither TIMP-1 nor TIMP-2 is required for the fibrotic response observed in the livers of schistosome-infected mice.

FIGURE 3.
FIGURE 3.

Liver histology of TIMP-1- and TIMP-2-deficient mice. Data are from several experiments at 9 and 14 wk after infection of C57BL/6 (□), TIMP-1 knockout (▵), and TIMP-2 knockout (▿) mice with 25 S. mansoni cercariae per mouse. The degree of liver fibrosis in TIMP-1 and -2-deficient mice is assessed by the amount of hydroxyproline in micromoles detected in liver per 10,000 eggs at 9 (A) and 14 wk (B) postinfection. Granuloma volume for TIMP-1 and -2-deficient mice was assessed by microscopy at 9 (C) and 14 wk (D). The average size of 30 granulomas is shown for each mouse. Mann-Whitney test p values vs C57BL/6 are shown. Each data point represents one mouse, while bars represent the median of each group.

Increased expression of several MMP in the livers of S. mansoni-infected mice

To determine whether specific MMP may be important in the fibrotic process, we examined the expression profiles of several MMP at multiple time points postinfection. All MMP studied were expressed constitutively at low levels before infection, but were significantly induced following the onset of egg laying (Fig. 4⇓). MMP-9 and -12 mRNA levels were modestly, but significantly, decreased at wk 3. By wk 6, their expression had returned to baseline levels, while MMP-13 was the only gene that showed significantly elevated expression at this early time point; by wk 9, however, levels of mRNA for all five MMP were significantly induced. MMP-2, -9, and -12 remained significantly elevated throughout the 16 wk of the study, while expression of MMP-3 and -13 steadily decreased after the acute stage of infection (wk 9). Thus, expression of MMP-2, -9, and -12 correlated well with the onset and progression of fibrosis.

FIGURE 4.
FIGURE 4.

Kinetics of MMP expression in infected C57BL/6 mice. Time course of liver mRNA expression for MMP-2 (A), MMP-3 (B), MMP-13 (C), MMP-9 (D), and MMP-12 (E) in S. mansoni-infected C57BL/6 mice. Expression values for each mouse have been normalized to the corresponding HPRT value. UI, uninfected mice. ∗, p < 0.05 vs uninfected C57BL/6 mice. Each data point represents one mouse, while bars represent the median value for each group.

IL-10/IFN-γ-deficient mice develop an exaggerated and highly polarized type 2 cytokine pattern that triggers a more severe fibrotic response

To better understand how ECM-related genes are regulated during a fibrotic disease and, in particular, to determine how type 2 cytokines influence their expression, we generated mice that were predicted to develop an exaggerated type 2 cytokine response. The cytokines IL-10 and IFN-γ are potent negative regulators of type 2 cytokine responses (40, 41, 42) and egg-induced immunopathology in schistosomiasis (43, 44). Therefore, we crossed IL-10- and IFN-γ-deficient animals to generate mice that were deficient in both mediators. The single- and double-deficient mice were infected with S. mansoni, and production of IL-4, IL-5, IL-10, IL-13, and IFN-γ was assessed in Ag-stimulated MLN cultures. Representative data from one of two separate experiments are shown in Fig. 5⇓. C57BL/6 and IFN-γ-deficient mice developed very similar responses, consistent with previous studies (45). MLN cells from both strains produced significant IL-4, IL-5, IL-10, and IL-13, but little or no IFN-γ following stimulation with SEA, SWAP, or Con A. In contrast, IL-10-deficient mice displayed a mixed phenotype, producing abundant IFN-γ and even greater amounts of the type-2 cytokines compared with WT mice. Notably, however, the double-cytokine-deficient mice developed an elevated Th2 response similar to IL-10-deficient mice, but unlike the IL-10-deficient animals, their response developed in the absence of IFN-γ. Thus, they were the only mice that manifested an exaggerated and highly polarized type 2 cytokine response.

FIGURE 5.
FIGURE 5.

Cytokine data from MLN cells of various cytokine-deficient mice showing the cytokine response of cultured primary MLN cells prepared from mice 8 wk after infection. Cells were cultured in medium alone or with SEA (20 μg/ml), SWAP (40 μg/ml), or Con A (1 μg/ml) for 72 h. The Th2-type cytokines, IL-4 (A), IL-5 (B), IL-13 (C), and IL-10 (E), and the Th1-type cytokine, IFN-γ (D), are shown. Error bars show variation between two separate experiments using 7–10 mice/group.

Liver fibrosis and granulomatous inflammation were also evaluated in the infected cytokine-deficient mice and directly compared with the TIMP and MMP mRNA responses. The data shown in Fig. 6⇓ are the combined results of two separate experiments. Fibrotic changes were evaluated by hydroxyproline assay, using liver samples collected at wk 8 postinfection. IFN-γ- and IL-10-deficient mice developed slightly increased levels of fibrosis compared with C57BL/6 mice (Fig. 6⇓A); however, the response in the IL-10/IFN-γ-deficient mice was significantly worse vs all groups, consistent with their exaggerated type 2 cytokine response. Of interest, granuloma size followed a slightly different pattern. Here, IL-10-deficient mice, which develop a mixed type 1/type 2 cytokine response, consistently developed the largest lesions (Fig. 6⇓B). Granuloma size also increased in the double-IL-10/IFN-γ-deficient mice, although this was not nearly as dramatic as in the IL-10-deficient animals. In contrast, granuloma size decreased slightly in the IFN-γ-deficient mice. Again, consistent with their increased type 2 cytokine response, the IL-10/IFN-γ-deficient mice showed a significant increase in granuloma eosinophils compared with C57BL/6 mice (p = 0.004; data not shown). The eosinophil response was not significantly different in the other cytokine knockouts (data not shown). It is also noteworthy that IL-10-deficient mice showed significant mortality by wk 8 postinfection, which prevented additional investigations at later time points (data not shown).

FIGURE 6.
FIGURE 6.

Liver histology of infected cytokine-deficient mice. Combined histologic data from two separate experiments at 8 wk after infection with 25 S. mansoni cercariae per mouse are shown. Each data point represents one mouse, while bars represent the median of each group. Mann-Whitney test p values vs C57BL/6 are shown. A, The degree of fibrosis is assessed by the amount of hydroxyproline in micromoles detected in liver per 10,000 eggs. B, Granuloma volume as assessed by microscopy. The average size of 30 granulomas is shown for each mouse.

Elevated fibrosis in IL-10/IFN-γ-deficient mice is associated with a distinct ECM-related gene expression profile

Liver RNA was isolated at wk 8 postinfection and analyzed by real-time RT-PCR to determine how fibrosis and type 2 cytokine dominance correlate with ECM-related gene expression. Representative data from one of two separate experiments are shown in Figs. 7⇓ and 8⇓. TIMP-1 (Fig. 7⇓A) and TIMP-2 (Fig. 7⇓B) mRNA were almost undetectable in uninfected mice. After infection, however, all four strains showed significant TIMP-1 and -2 mRNA responses. Compared with C57BL/6 mice, responses in IFN-γ-deficient mice were not significantly different, whereas the IL-10/IFN-γ-deficient strain showed a marked decrease in both TIMP-1 and -2 mRNA. TIMP-2 expression was also significantly decreased in the IL-10-deficient mice, suggesting that IL-10 may be important for TIMP-2 mRNA expression in vivo. Consistent with previous observations, expression of TIMP-3 did not consistently change following infection (data not shown). TIMP-4 was not analyzed, because earlier studies showed little TIMP-4 mRNA expression in the liver of uninfected or infected mice.

FIGURE 7.
FIGURE 7.

Liver mRNA expression for TIMP-1 (A) and TIMP-2 (B) in mice infected with S. mansoni for 8 wk. ▵, C57BL/6 mice; ----, IFN-γ-deficient mice; ⋄, IL-10 knockout mice; ○, IL-10/IFN-γ-deficient mice. Filled shapes represent infected mice; open shapes represent uninfected mice (UI). Expression values for each mouse have been normalized to the corresponding HPRT value. ∗, p < 0.05 vs C57BL/6. Each data point represents one mouse, while bars represent the median value for each group.

FIGURE 8.
FIGURE 8.

Liver mRNA expression for MMP-2 (A), MMP-3 (B), MMP-13 (C), MMP-12 (D), and MMP-9 (E) in mice infected with S. mansoni for 8 wk. ▵, C57BL/6 mice; ▿, IFN-γ-deficient mice; ⋄, IL-10 knockout mice; ○, IL-10/IFN-γ-deficient mice. Filled shapes represent infected mice, open shapes represent uninfected mice (UI). Expression values for each mouse have been normalized to the corresponding HPRT value. ∗, p < 0.05 vs C57BL/6. Each data point represents one mouse, while bars represent the median value for each group.

MMP mRNA expression was minimal in uninfected mice, whereas significant induction of all five MMP was seen following infection (Fig. 8⇑). A similar MMP-2 mRNA response was seen among the C57BL/6 and IFN-γ-deficient, and IL-10/IFN-γ-deficient mice (Fig. 8⇑A); however, MMP-2 levels in IL-10-deficient mice decreased significantly from those in C57BL/6 mice. MMP-3 mRNA levels were similar between C57BL/6 and IFN-γ-deficient mice, although expression variably increased in the IL-10-deficient mice and decreased significantly in the combined absence of IFN-γ and IL-10 (Fig. 8⇑B). MMP-13 levels were similarly elevated in the C57BL/6 and IFN-γ-deficient mice, while expression varied widely in the IL-10-deficient animals. The double-deficient mice, however, displayed a significant decrease (Fig. 8⇑C). MMP-12 results showed no significant change in the absence of IFN-γ, but expression was significantly reduced in both the IL-10- and IL-10/IFN-γ-deficient mice (Fig. 8⇑D). In contrast to the other MMP studied, the 92-kDa gelatinase MMP-9 was regulated in a distinct manner. Although expression of MMP-9 increased in all four strains following infection, there was a marked and significant increase in the double IL-10/IFN-γ-deficient mice. Notably, these animals were the group that showed the greatest increase in liver fibrosis following infection (Fig. 4⇑A).

ECM accumulation or removal probably results from changes in both TIMP and MMP expression (38, 46). Indeed, the anti-MMP activity of TIMP should be exaggerated by a coincidental decrease in MMP expression, and vice versa, either of which may affect net MMP activity. Therefore, we analyzed the ratios of MMP to TIMP mRNA expression to better evaluate how changes in gene expression relate to fibrogenesis in the various cytokine knockout mice (Table II⇓). For these analyses, the median TIMP-1 and MMP mRNA values for each knockout group were divided by the corresponding WT values. The normalized MMP values were then divided by the calculated TIMP-1 result to determine whether there was a relative difference in MMP expression. Ratios greater than 1 denote a relative abundance of MMP message compared with TIMP-1 in the knockout vs WT mice. Likewise, a number less than 1 would be consistent with a relative increase in TIMP-1 mRNA expression.

View this table:
Table II.

Ratios of normalized MMP mRNA expression to normalized TIMP-1 mRNA expressiona

For MMP-12 and -13, the ratios were equivalent among the groups, which suggests these MMP are unlikely to explain the differences in fibrosis observed in the cytokine-deficient mice. The IFN-γ-deficient and IL-10/IFN-γ-deficient mice also displayed approximately equivalent values for MMP-3. There was, however, an increase in the MMP-3:TIMP-1 ratio in the IL-10-deficient mice. The ratios for MMP-2 and MMP-9 to TIMP-1 were elevated in the IL-10/IFN-γ-deficient mice, while the IL-10-deficient mice displayed a more modest increase only in MMP-9. Comparable results were obtained when TIMP-2 was substituted for TIMP-1 in the same calculations (data not shown). Together these data illustrate that the development of severe liver fibrosis in infected IL-10/IFN-γ-deficient mice is most closely associated with increased MMP-2/MMP-9 and decreased TIMP-1/TIMP-2 mRNA expression, which was contrary to expectations.

Discussion

Schistosomes are a significant worldwide cause of morbidity and mortality, mainly through their induction of liver fibrosis and resulting sequelae. Many upstream aspects of the fibrotic response in murine schistosomiasis, such as the importance of Th2 cells (47) and specific effector cytokines such as IL-4, IL-10, and IL-13 have been well characterized (5, 6, 7); however, the downstream role of ECM-modifying genes has not been examined in detail. Because TIMP have been purported to be necessary for fibrogenesis, we first examined their role in this process. Additionally, there is little information about how type 1/type 2 cytokines regulate the expression of ECM genes. Therefore, we also determined whether Th2-mediated liver fibrosis is associated with specific patterns of MMP and TIMP mRNA expression.

In this disease damage to the liver begins ∼5 wk following infection, when paired adult parasites begin laying eggs. Eggs are continuously deposited in the liver, inducing a chronic granulomatous inflammatory response. Significant fibrotic changes are observed by wk 7 and continue to increase in a linear fashion throughout the infection (44). Consistent with this pattern of fibrosis, we observed little change in TIMP mRNA expression before the onset of egg laying; however, marked changes were observed after wk 6, and expression of TIMP-1 and -2 was greatly elevated by wk 9, when the size of granulomas has also reached a maximum (48). Strikingly, the initial pattern of expression of TIMP-1 and -2 mRNA in the liver closely mirrored the appearance of pathology, suggesting that these genes might indeed be playing an important role. Expression of both genes decreased rapidly in the chronic stage of infection, however, with TIMP-2 expression returning to baseline levels by wk 16. These observations suggested that the regulatory effects of TIMP-1 and -2 might be restricted to the acute phase of infection. The data were unexpected, because liver collagen content more than doubles between wk 9 and 16 (see Fig. 1⇑A). Thus, TIMP-1/TIMP-2 mRNAs are decreasing at a time when fibrosis is markedly increasing.

Consistent with the above, our studies with TIMP-1- and TIMP-2-deficient mice demonstrated that TIMP-1 and -2 play little or no regulatory role in the pathogenesis of murine schistosomiasis. TIMP-1-deficient and WT mice developed similar levels of fibrosis at the acute and chronic stages postinfection. Thus, in contrast to what might have been predicted there was no evidence of a profibrogenic role for TIMP-1. Giemsa-stained liver sections also revealed no significant change in the granulomatous response. Similar findings were generated with TIMP-2-deficient mice. In fact, the TIMP-2 animals actually manifested a slight increase in fibrosis at the acute stage postinfection, which may have been attributable to the general decrease in MMP expression observed in these animals at this time point (Fig. 2⇑B). It is possible, however, that TIMP-1 and/or TIMP-2 are important regulators of fibrosis in other tissues or in other fibrotic disorders. Nevertheless, the fact that development of renal interstitial fibrosis was also unaffected by TIMP-1 deficiency suggests these findings may be more globally applicable (49). It is also important to mention that other ECM-regulating genes, such as α1-macroglobulin or plasminogen activator inhibitor 1, could be compensating for the absence of TIMP-1 and TIMP-2, which may provide an alternative explanation for our findings.

Changes in MMP expression could also affect the development of fibrosis. We therefore examined the liver MMP response during infection and found that expression of some MMP varied in a manner consistent with the development of fibrotic pathology. To help identify those MMP that might be important, we used cytokine-deficient mice that generate a stronger Th2 response and more severe fibrosis than WT mice following infection. Not unlike our initial kinetic studies, these experiments were designed to determine whether specific changes in MMP gene expression were predictive of the degree of fibrosis. As predicted, the IL-10/IFN-γ-deficient mice developed an exaggerated Th2 response and more severe fibrosis than did mice in the other three groups. These findings formally document the combined anti-fibrotic roles of IL-10 and IFN-γ in the schistosomiasis model. Of interest, while fibrosis was more severe in the double knockout mice, the IL-10-deficient animals consistently developed the largest granulomas. Thus, worsening fibrosis does not necessarily correlate with an increasing inflammatory response but does strongly correlate with the relative dominance of type 2 cytokine expression. These observations suggest that IFN-γ exhibits distinct functional activities in this disease by both enhancing granulomatous inflammation and simultaneously decreasing collagen deposition. Because of their unique immunologic and pathologic responses, these different cytokine-deficient mice provide excellent tools to investigate how changes in type 1/type 2 cytokine expression regulate ECM gene expression in vivo. In this regard, we found markedly decreased expression of TIMP-1 mRNA in the livers of the type 2-polarized IL-10/IFN-γ-deficient mice as well as decreased TIMP-2 in the two strains lacking IL-10. These data are entirely consistent with our TIMP-1 and -2 knockout experiments, because they reveal a negative correlation between TIMP expression and development of fibrosis.

Surprisingly, the data generated with the cytokine knockout mice showed that MMP-2 and -9 expression increased in comparison to TIMP-1 and -2 in the more fibrotic IL-10/IFN-γ-deficient mice. Notably, WT C57BL/6 mice exhibited a similar expression pattern at 16 wk postinfection, when fibrotic changes in the liver were reaching a maximum (Figs. 1⇑ and 4⇑). Indeed, TIMP-1 and -2 mRNA expression at 16 wk had decreased from the peak response observed on wk 12 and 9, respectively, while expression of MMP-2 and -9 remained relatively unchanged during this period. Thus, the kinetic data as well as the cytokine knockout studies suggest that increasing levels of fibrosis correlate with higher MMP-2/9:TIMP-1/2 ratios.

Consistent with our work, others have demonstrated that elevated MMP-2 expression is associated with hepatic fibrosis in CCl4-treated mice (50); however, that work also showed that production of MMP-2 occurs during recovery from CCl4-induced hepatic fibrosis. Therefore, its role in the regulation of fibrosis remains unclear. In the liver, MMP-2 is produced abundantly by activated hepatic stellate cell (HSC) and fibroblasts, although other resident liver cells may be minor producers of MMP-2 (51). This MMP is up-regulated in cultured rat HSC upon migration, and other studies suggest that MMP-2 is involved in the proliferation of HSC (52, 53). Thus, because MMP-2 may facilitate cell movement and proliferation, its increased expression may be important in executing the HSC programmed response to inflammatory insult. However, the importance of MMP-2 in fibrogenesis is brought into question by recent work in TIMP-2-deficient mice. These mice show a defect in pro-MMP-2 activation in several experimental systems (54). Therefore, given that our TIMP-2-deficient mice should also manifest a deficiency in active MMP-2, this metalloprotease may not be vital to the regulation of fibrosis.

In contrast to MMP-2, MMP-9 is widely produced by many cell types, including hepatocytes, Kupffer cells, neutrophils, eosinophils, and T cells (55, 56, 57, 58). One group has postulated a possible connection between MMP-9 and fibrosis by showing that MMP-9-deficient mice had reduced fibrosis as well as decreased neutrophil and macrophage infiltration in resolving cardiac infarcts (59). Expression of MMP-9 by inflammatory cells such as neutrophils, eosinophils, and T cells may facilitate their movement across the basement membrane (57, 58, 60). Thus, its role in fibrosis may be indirect, by recruiting cells that more directly orchestrate the fibrotic response. The requirement of MMP-9 for extravasation has, however, been called into question in other studies (61, 62). MMP-9 expression is also closely associated with hepatocyte regeneration after liver injury; thus, like MMP-2, MMP-9 may be involved in the recovery phase (63). It is also possible that expression of both genes is up-regulated to limit or control the overall magnitude of fibrosis as the infection becomes chronic. Finally, both MMP-2 and -9 are believed to be important in angiogenesis (64). Because portal circulation may be disrupted in the fibrotic IL-10/IFN-γ-deficient mice, the expression of MMP-2 and -9 may increase to facilitate the formation of collateral circulation to bypass these blockages (65). It has been previously described that significant vascular remodeling and neovascularization occur in the livers of schistosome-infected mice, which would support this hypothesis (66).

It has been widely suggested that TIMP, via their MMP inhibitory activity, would be important for fibrogenesis. In support of this hypothesis, a mouse that overexpresses human TIMP-1 was shown to develop more severe liver fibrosis when exposed to CCl4 (39); however, other recent studies in addition to our work question the paradigm that fibrosis is prevented by MMP activity and promoted by TIMP (49, 67). Indeed, the use of a synthetic MMP inhibitor batimastat was recently shown to decrease fibrosis in a bleomycin-induced pulmonary fibrosis model (67). Given these conflicting results, further testing in a variety of in vivo models will be needed before we attain a full understanding of the specific functions of MMP and TIMP in fibrogenesis. In summary, these data highlight the important combined inhibitory roles played by IFN-γ and IL-10 in schistosome egg-induced liver fibrosis and demonstrate that development of more severe fibrosis in the absence of these cytokines is most closely associated with increases in MMP-2 and MMP-9. Moreover, these studies clearly demonstrate no essential role for TIMP-1 or -2 in the pathogenesis of schistosomiasis.

Acknowledgments

We thank Drs. Matthias Hesse, Alan Sher, Margaret Mentink, and Mary Leusink for critically reviewing this manuscript. We also thank Dr. Fred Lewis and Chris Rowe at the Biomedical Research Institute for providing the parasites used in this study.

Footnotes

  • ↵1 B.V. is a Howard Hughes Medical Institute-National Institutes of Health Research Scholar.

  • ↵2 Address correspondence and reprint requests to Dr. Thomas A. Wynn, Schistosomiasis Immunology and Pathology Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Drive, Room 4/126, Bethesda, MD 20892. E-mail address: twynn{at}niaid.nih.gov

  • ↵3 Abbreviations used in this paper: ECM, extracellular matrix; HPRT, hypoxanthine phosphoribosyltransferase; HSC, hepatic stellate cell; MLN, mesenteric lymph node; MMP, matrix metalloprotease; SEA, soluble S. mansoni egg Ag; SWAP, soluble worm Ag preparation; TIMP, tissue inhibitor of metalloprotease; WT, wild type.

  • Received June 20, 2001.
  • Accepted October 9, 2001.

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