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Matrilysins-1 and -2 (MMP-7 and -26) and Metalloelastase (MMP-12), Unlike MMP-19, are Up-Regulated in Necrotizing Enterocolitis

Bister, Ville*; Salmela, Mikko T.*; Heikkilä, Päivi†; Anttila, Annaleena‡; Rintala, Risto‡; Isaka, Keichi§; Andersson, Sture‡; Saarialho-Kere, Ulpu*¶

Journal of Pediatric Gastroenterology & Nutrition: January 2005 - Volume 40 - Issue 1 - pp 60-66
Original Articles: Gastroenterology

Objectives: Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants characterized histologically by extensive tissue injury and inflammation. Matrix metalloproteinases (MMP) are involved in tissue remodeling and cell migration, both being important aspects of inflammatory disease. The aim of this study was to investigate whether MMPs play a role in the pathogenesis of NEC.

Methods: Expression of MMP-1, -7, -9, -10, -12, -19 and -26 was studied using in situ hybridization/immunohistochemistry in samples intestinal tissue removed from 15 patients with NEC; in 7 of them control samples were obtained at closure of stomas. Six intestinal samples from patients with intestinal atresia and four samples of necrosis were also included in the material examined. Laminin-5 was immunostained to find migrating enterocytes and cytokeratin to delineate mucosal epithelium.

Results: MMP-7 protein was upregulated in the epithelium of 12/18 NEC samples. MMP-26 was induced in stromal cells of 12/17 NEC specimens. Stromal expression was found for MMP-1 and -12 mRNAs in 7/18 samples. MMP-1 was also detected in the epithelium of regenerating areas. Both NEC and stoma samples expressed MMP-9 in inflammatory cells. Epithelial MMP-19 was downregulated in NEC.

Conclusions: Our results suggest that several MMPs may be major factors in tissue destruction and remodeling in NEC. Targeted inhibition of matrilysins, using synthetic MMP inhibitors or blockers of their signal transduction pathways, may represent a novel therapeutic option for the treatment of intestinal inflammation associated with NEC.

Departments of *Dermatology and †Pathology, Helsinki University Central Hospital; ‡Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland; §Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan; and ¶Department of Dermatology, Karolinska Institutet at Stockholm Soder Hospital, Stockholm, Sweden

Received February 10, 2004; accepted August 16, 2004.

Address all correspondence and requests for reprints to Dr. Ulpu Saarialho-Kere, Department of Dermatology, Karolinska Institutet, Stockholm Soder Hospital, SE-11883 Stockholm, Sweden (e-mail:

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Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants with an overall mortality rate of 20 to 30% (1,2). Although several predisposing factors have been identified, such as prematurity, enteral feeding and infection, its ultimate pathobiology is still unknown (3). The symptoms vary dramatically from benign gastrointestinal disturbance to intestinal gangrene, perforation, sepsis and shock (2,4). Microscopically coagulative and haemorrhagic necrosis, limited to the mucosa in early stages, but at least focally transmural when the surgical removal occurs, are observed in the ileum and caecum (2). Mixed intestinal bacteria are often visible in the lumen or the necrotic superficial mucosa.

The matrix metalloproteinases (MMPs) constitute a superfamily of 23 human zinc-dependent endopeptidases classified according to their structure and substrate specificity into six subgroups: collagenases, gelatinases, stromelysin-like MMPs, matrilysins, membrane-type MMPs and other MMPs (5). As a group they can degrade all components of the extracellular matrix. However, they also regulate cellular growth factor responses and inflammatory reactions by cleaving and releasing growth factors, cytokines, chemokines, and their receptors (6,7). The expression of various MMPs, such as stromelysin-1 and -2 and metalloelastase, has been implicated as one of the main factors contributing to mucosal ulceration in inflammatory bowel disease (IBD) (8-10).

MMPs are involved in tissue remodeling and cell migration, which are the most important aspects of inflammatory disease. Subepithelial stromal cells cross-talk with enterocytes and the whole inflammatory process is governed by signals produced by altered cell-cell and cell-matrix interactions. Inhibition of MMPs, either by synthetic MMP inhibitors or by interfering with their signal transduction pathways, represents a novel therapeutic approach for treatment of intestinal inflammation (5,11,12). The aim of this study was to investigate the contribution of several MMPs to the pathogenesis of NEC.

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Formalin-fixed paraffin-embedded peroperative specimens of NEC (n = 18), atresia (n = 6) and necrosis (n = 4), removed for clinical reasons and for routine histopathology, were obtained from the Departments of Pathology and Pediatrics, Helsinki University Central Hospital, Finland. Also the corresponding stomas of seven NEC patients were studied as controls. Clinical information on the patients is presented in Table 1.

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Immunohistochemistry was performed using the avidin-biotin-peroxidase complex technique (DAKO and Vector rabbit). Diaminobenzidine (DAB) was used as chromogenic substrate and sections were counterstained with Mayer's haematoxylin. Samples were immunostained using polyclonal antibodies to MMP-19 (PC 374; Oncogene Research Products, Cambridge, MA) and MMP-26 (13), as well as using monoclonal antibodies to MMP-7 (IM 40L, Calbiochem, Cambridge, MA) and MMP-9 (PC 213; Oncogene Research Products). The cells producing laminin-5 were detected with polyclonal antibodies against the gamma-2 chain. Laminin-5 and MMP-9 stainings required pretreatment with 10 mg/ml trypsin. Anti-MMP-19 antibodies were diluted 1:50 to 1:70, MMP-26 1:100 to 200, MMP-7 1:80, MMP-9 1:75 and laminin-5 1:700. MMP-19, -7 and laminin-5 antibodies were reacted overnight at +4 C. MMP-26 was incubated at room temperature for 1 h, and MMP-9 in +37°C for 30 minutes. Epithelial areas were identified by immunostaining for cytokeratin CAM 5.2 1:75 (Becton-Dickinson, B-9320 Erembodegem-Aalst, Belgium). Negative controls included rabbit pre-immune serum (MMP-26) or rabbit IgG (MMP-19). The specificity of MMP-19 staining has been previously demonstrated by us (14). Positive controls used were samples of breast cancer (MMP-19), placenta and endometrium (MMP-26).

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In Situ Hybridisation

The production and specificity of MMP-1 (15,16), MMP-10 (8,17), MMP-7 (15) and MMP-12 (8) probes have previously been described. Afer deparaffinization and rehydration 5 μm sections were pretreated with proteinase K (1 mg/ml) and washed in 0,1 M triethanolamine containing 0.25% acetic anhydride. Sections were hybridized overnight at 50 to 55°C with 35S-labeled probes and washed thereafter under stringent conditions and treated with RNAase A to remove unhybridized probe. After 20 to 40 days autoradiographic exposure, the photographic emulsion was developed and the slides were stained with Mayer's haematoxylin. Previously positive samples were used as controls: ulcerative colitis (UC) for MMPs-1, -7 and -12 and acute cutaneous wounds for MMP-10.

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Collagenase-1 MMP-1 mRNA was expressed in 8/18 NEC samples in mesenchymal fibroblast-like cells, correlating with the presence of acute necrosis (Fig. 1A; Table 2). MMP-1 was also detected in the epithelium of five NEC samples and that of three stomas in regenerative areas (Fig. 1B, C). Also, 2/7 control stomas, 2/6 samples of atresia and 2/4 samples of necrosis were also positive for stromal MMP-1 mRNA (Table 2).

Stromelysin-2 MMP-10 was expressed by fibroblast-like cells in the stroma around the crypts in 3/18 NEC specimens only. Expression was found in mesenchymal cells in acute necrosis and regenerative areas (Table 2). Also, 4/7 of stomas were positive for MMP-10 (Table 2).

92 kDa gelatinase MMP-9 expression was detected in neutrophils in the majority of NEC, stoma and atresia samples (Table 2; Fig. 1D). MMP-9 positive macrophages were also detected in a subpopulation of all diagnostic categories examined (Fig. 1D) in the areas of serosa and neutrophil accumulations. Control stomas had the same expression of MMP-9 as the original NEC sample from the same patient, but the positive staining concentrated also in the submucosa in the cutting edge of the stoma (data not shown).

Metalloelastase MMP-12 was expressed by macrophages inside the villi and in the stroma underlining the crypts in 7/18 NEC specimens (Fig. 2A, B). Also, 2/7 of stomas were positive (data not shown). Overall expression in all NEC samples was more intense than in the positive stomas. Atresia samples were negative for MMP-12, reflecting the absence of widespread inflammation in these samples, whereas 2/4 samples with necrosis were positive.

Matrilysin-1 MMP-7 expression was most intensive in injured epithelium of 12/17 NEC samples (Fig. 3A; Table 2), but the intensity decreased towards the necrotic areas. MMP-7 was also detected in regenerating mucosal epithelium in both NEC and necrosis specimens (Fig. 3B). Positive epithelial cells were also found in atresia samples (2/6), in necrotic ones (3/4) and in control stomas (3/7). However, the number of positive cells did not equal that of NEC samples.

Matrilysin-2 MMP-26 was expressed in the fairly intact villous epithelium of ileum (Fig. 3C). In NEC samples, its expression was downregulated in the epithelium of injured, inflamed areas (Fig. 3D). Also, stromal cells were positive for MMP-26 in NEC, especially macrophages in the regenerative areas (Fig. 3D, E). Also the vascular endothelium showed positive staining in several samples (Fig. 3F). In stoma samples, the mucosal epithelium was generally negative and stromal signal was only occasionally detected unlike in NEC.

MMP-19 Only 5/18 NEC samples had positive immunosignal for MMP-19. MMP-19 protein was then detected in the crypt epithelium (Fig. 2C). All stoma samples displayed epithelial expression of MMP-19 (Fig. 2D). The overall expression in the stoma samples was more intense and even than in NEC samples.

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NEC is a major cause of morbidity and mortality in preterm infants. Together prematurity, enteral feeding and bacterial colonization have been hypothesized to result in an exaggerated inflammatory response, leading to ischemic bowel necrosis (18). While this study was in progress, Pender et al. (19) reported increased expression for MMP-3 in myofibroblasts of NEC patients in vivo, suggesting that MMP-3, but not MMP-1 or -9, is responsible for the extensive tissue injury seen in NEC. Our study extends and partly confirms their findings. The differences between our two studies may be because our control samples were obtained from patients age matched to the patient with NEC, while patients and controls significantly differed in the study of Pender et al. (19) (10 d vs. 4-5 mo). Furthermore, our major focus was on the contribution of more novel MMPs to NEC-associated tissue changes. Since MMPs probably act in a cascade-like fashion as their function is regulated by activation by plasmin, furin or other MMPs, and by specific tissue inhibitors of MMPs, TIMPs (20), we considered it important to investigate the contribution of several MMPs to NEC simultaneously.

A number of studies suggest that MMPs are the most important group of proteolytic enzymes responsible for the breakdown of extracellular matrix in ulcerative colitis, another disorder characterized by intestinal tissue destruction (8-10,21,22). MMP-1 was detected in more than half of our NEC samples in fibroblast-like cells of the stroma. In IBD, MMP-1 is involved in mucosal destruction (11,23) and our stromal MMP-1 expression may well reflect tissue injury in NEC. Interestingly, MMP-1 was also detected in the epithelial cells of regenerating areas, unlike in IBD (10,15,24), and this agrees with our novel results on ischemic colitis (25).

MMP-9, primarily an inflammatory cell-derived gelatinase, is a major factor in adult intestinal tissue destruction and inflammation (21,26). Studies in rats have demonstrated that colonic obstruction and trauma up-regulate gelatinases and decrease collagen concentration in the colonic wall (27). Hence, obstruction and local trauma may be of significance in upregulating leukocyte MMP-9 activity in atresia samples (Table 2).

MMP-10 is a potent activator of proMMP-1. It was rarely noted in stromal cells in NEC and was absent from epithelial cells. In an experimental model of IBD, MMP-10 was expressed in areas with most severe injury (9) and stromal expression in NEC probably reflects the same phenomenon.

Expression of MMP-12 mRNA was detected in macrophages in a subset of NEC samples. TNF-α and IL-1β upregulate MMP-12 in macrophages in vitro (28). MMP-12 may help macrophages to migrate to sites of inflammation by degrading BMs of endothelial cells and that of mucosal epithelium. MMP-12 may also regulate inflammatory response since it can activate TNF-α (29).

In this study, we showed that MMP-7 is expressed in NEC, but not uniformly in control stoma tissue, suggesting that it is of significance in tissue destruction in NEC. Expression of MMP-7 has been reported to correlate with the degree of inflammation in UC (10). In the small intestine MMP-7 functions in host-defense by activating defensins (30). Bacterial exposure is a potent signal regulating MMP-7 expression in epithelial cells (31) and may also be the mechanism for its induction in NEC. Epithelial disruption is needed for MMP-7 induction in human intestinal epithelial cells in vivo (10,15). MMP-7 serves key functions in both epithelial defense and repair (30).

MMP-26 (matrilysin-2, endometase), the smallest MMP, participates in enterocyte migration over ulcerated areas in IBD (32). We detected MMP-26 also in endothelial cells, agreeing with a recent report on endometrial cancer (33). Upregulation of MMP-26 in the stroma of NEC samples was marked and, to our knowledge, this is the first work describing MMP-26 protein in macrophages.

MMP-19 has been found in fibroblasts, myoepithelial and smooth muscle cells as well as associated with the cell surface of myeloid cells (34,35). The absence of MMP-19 from the majority of NEC samples with severe tissue injury may reflect the fact that it has previously been implicated in normal tissue turnover (34). We have recently detected MMP-19 in hyperproliferating keratinocytes (14) and thus its presence particularly in the cryptal epithelium of stomas may be associated with enterocyte proliferation and regeneration.

Increased levels of TNF-α and decreased levels of IFN-γ have been noted in vivo in human NEC (19). In experimental models TNF-α is known to cause tissue injury by stimulating mucosal mesenchymal cells to secrete MMPs and TNF-α antibodies can block this cascade (12). TNF-α can upregulate several MMPs, such as MMP-9 and MMP-12 in macrophages (36,37), MMP-19 in epithelial cells (14), and MMP-1, -10 and -7 in Caco-2, WiDR and HT-29 cell cultures (25). TNF-α exposure to myofibroblasts induces MMP-1 and -3 secretion (38,39). TNF-α also downregulates TIMP-1 expression (40), which favors matrix breakdown. Thus, it seems that TNF-α may well be the crucial cytokine upregulating MMPs that induces tissue destruction in NEC.

In conclusion, our results suggest that several other MMPs in addition to MMP-3 may be factors in tissue destruction and remodeling in NEC. Targeted inhibition of MMP-7 not only in UC (10), but also in NEC, may represent a novel therapeutic option for the treatment of intestinal inflammation.

Acknowledgments: The authors thank Mrs. Alli Tallqvist for skillful technical assistance. This study was supported by grants from the Academy of Finland, Sigrid Jusélius Foundation, Finska Läkaresällskapet, and Helsinki University Central Hospital Research Funds (EVO).

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Collagenase; Immunohistochemistry; Gut injury; Enterocyte

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