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* Division of Infection, Inflammation and Repair, University of Southampton School of Medicine, Southampton, United Kingdom;
Merck Research Laboratories, Rahway, NJ; and
Department of Biochemistry, Centre for Molecular Microbiology and Infection, Imperial College, South Kensington, United Kingdom
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–/– mice, and here we show that this is associated with an increase in stromelysin-1 (MMP3) transcripts in colonic tissues. We have therefore investigated the role of MMP3 in colonic mucosal hyperplasia and the local Th1 responses using MMP3–/– mice. In MMP3–/– mice, similar mucosal thickening was observed after infection as in wild-type (WT) mice. Colonic tissues from MMP3–/– mice showed a compensatory increase in the expression of other MMP transcripts, such as MMP7 and MMP12. However, MMP3–/– mice showed delayed clearance of bacteria and delayed appearance of CD4+ T lymphocytes into intestinal lamina propria. CSFE-labeled mesenteric lymph node CD4+ T lymphocytes from infected WT mice migrated in fewer numbers into the mesenteric lymph nodes and colon of MMP3–/– mice than into those of WT mice. These studies show that mucosal remodeling can occur in the absence of MMP3, but that MMP3 plays a role in the migration of CD4+ T lymphocytes to the intestinal mucosa. |
Introduction |
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, and IFN- (3, 4).
To elucidate the role of proinflammatory cytokines in host defense and mucosal pathology in vivo, mice deficient in TNF-, IFN-, and IL-12 were orally infected with live C. rodentium (5, 6). These animals showed various degrees of impairment in their ability to clear infection, but remarkably, mucosal pathology was greater in these mice than in wild-type (WT) controls.
Matrix metalloproteinases (MMPs) are a family of proteases important in the turnover of extracellular matrix and cell migration (reviewed in Ref. 7). MMPs belong to the matrixin subfamily of the metzincin superfamily of metalloproteinases. To date, >23 different MMPs have been cloned, and additional members continue to be identified. Structurally, all MMPs share a similar prodomain and catalytic domain, which act on a broad spectrum of the extracellular matrix components. Most MMPs are secreted as proenzymes and require proteolytic cleavage for activation. Inhibition of MMPs is conducted by endogenous MMP inhibitors, tissue inhibitor of metalloproteinases (TIMP), which contain four members (TIMP1–4). MMPs have been implicated in tissue injury and matrix remodeling in various chronic inflammatory diseases such as IBD, asthma, and rheumatoid arthritis. In the intestine, MMPs are highly expressed at ulcer edges in patients with chronic IBD and play a crucial role in the tissue injury that follows T cell activation in explants of human fetal small bowel (8, 9, 10). In celiac disease, where a mucosal Th1 response to gluten drives mucosal remodeling and growth, MMP3 is overexpressed in fibroblasts immediately under the epithelium (11).
Taken together, these results suggest that MMPs, especially MMP3, is involved in immune-mediated tissue injury in the intestine. MMPs probably play a role in degrading the matrix to produce mucosal ulceration, but they may also be important in controlling matrix turnover and in the mucosal thickening seen in experimental and clinical intestinal inflammation.
In this work, therefore, we first examined MMP3 in the colon of WT and TNF-, IFN-, and IL-12 knockout (KO) mice infected with C. rodentium and subsequently investigated mucosal hyperplasia and the local immune responses in MMP3null mice.
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Materials and Methods |
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Female 6- to 8-wk-old C57BL/6J and B10R111 mice were purchased from Harlan Olac (Bichester, U.K.) or Bantin and Kingman Universal (Hull, U.K.). TNFRp55–/–, IL-12p40–/–, and IFN-–/– mice (backcrossed to C57BL/6 background at least 10 times) were originally purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained by homozygous matings under contract at Bantin and Kingman Universal. MMP3–/– mice were obtained from John Mudgett, Merck Research Laboratories (Rahway, NJ) and were maintained by homozygous matings. All mice came from specific pathogen-free colonies. They were kept in specific pathogen-free environment with free access to sterilized food and water. All experiments used five to six mice per group and were repeated at least twice.
Bacterial strains and oral infection of mice
A nalidixic acid-resistant isolate of C. rodentium (formerly Citrobacter freundii biotype 4280) was used in this study. DBS255 (pCVD438) is a C. rodentium eae (intimin ) strain, which expresses biologically active intimin and is virulent in mice (12). Bacterial inocula were prepared by culturing bacteria overnight at 37°C in 10 ml of Luria broth containing nalidixic acid (100 µg/ml) plus chloramphenicol (50 µg/ml). Cultures were harvested by centrifugation and resuspended in 0.1 volume of PBS. Mice were orally inoculated with 200 µl of the bacterial suspension with a gavage needle. The viable count of the inoculum was determined by plating on Luria-Bertani agar containing appropriate antibiotics. In all experiments, mice received 1–4 x 109 CFU.
Measurement of pathogen burden
At selected times postinfection, mice were killed by cervical dislocation. The terminal 4 cm of the colon were removed, and the colon was weighed after removal of fecal pellets. In some experiments, 1-cm samples of distal colon were snap-frozen in liquid nitrogen for subsequent immunohistological analysis, cytokine RT-PCR, and MMP Western blotting. Spleens and colons were homogenized mechanically using a Seward 80 stomacher (Seward Medica, London, U.K.). The number of viable bacteria in organ homogenates was determined by viable count on Luria-Bertani agar containing 100 µg/ml naladixic acid and 50 µg/ml chloramphenicol. The limit of sensitivity was 10 CFU/organ.
Immunohistochemistry and measurement of mucosal thickness
A three-step avidin-peroxidase staining was performed on 5-µm frozen sections as described previously (5) using mABs YTS191 (anti-CD4), anti-
7 and anti-mouse MAdCAM (both from BD BioSciences, Oxford, U.K.). Biotin-conjugated rabbit anti-rat IgG (1/50) and avidin peroxidase (1/200; DAKO, Cambridge, U.K.) were diluted in Tris-buffered saline (pH 7.6) containing 4% (v/v) normal mouse serum (Harlan Seralab, Oxon, U.K.). Peroxidase activity was detected with 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, Poole, U.K.) in 0.5 mg/ml in Tris-HCl (pH 7.6), containing 0.01% H2O2 (Sigma-Aldrich). The density of positive cells in the lamina propria was determined by image analysis (Carl Zeiss, Welwyn Garden City, Hertfordshire, U.K.). Mucosal thickness was measured by micrometry on H&E-stained sections, with 10 measurements being taken in the distal colons of individual mice (five to six mice/group). Only well-oriented pieces of tissues were analyzed.
Analysis of humoral immune responses
On day 21 postinfection, the animals were killed, and blood samples were collected by cardiac puncture. The serum was collected and stored at –20°C until it was analyzed. For analysis of Ag-specific Ab responses, wells of microtiter plates (Maxisorb plates; Nunc, Naperville, IL) were coated overnight at 4°C with 100 µl of a bicarbonate solution (pH 9.6) containing recombinant EspA (2.5 µg/ml). After a washing with PBS with Tween 20 (0.05% v/v), wells were blocked by addition of 1.5% (w/v) BSA in PBS for 1 h. Plates were then washed twice with PBS-Tween 20 before sera from individual mice were added and serially diluted in PBS-Tween 20 containing 0.2% (w/v) BSA and incubated for 2 h at 37°C. For the determination of isotype-specific Ab titers, wells were washed with PBS-Tween 20 before addition of 100 µl of an Ig-specific HRP conjugate (DAKO, High Wycombe, U.K.) diluted 1/1000 in PBS-Tween 20 containing 0.2% (w/v) BSA for 2 h at 37°C. Finally, after a washing with PBS-Tween 20, bound Ab was detected by addition of o-phenylenediamine substrate (Sigma-Aldrich) and the OD490 was measured. Titers were determined arbitrarily as the reciprocal of the serum dilution corresponding to an OD of 0.3.
Protein extraction
Colonic samples (2 cm) were snap-frozen at –70°C and homogenized in ice-cold extraction buffer (50 mM Tris-HCl (pH 7.4), 10 mM CaCl2, 0.05% Brij 35, 0.25% Triton X-100) at maximum speed for 30 s using an IKA tissue homogenizer (Fisher Scientific, Loughborough, U.K.). The homogenates were centrifuged at 13,000 rpm for 10 min at 4°C, and the supernatants were removed and assayed for protein concentration (Bio-Rad, Hemel Hempstead, Hertfordshire, U.K.). Homogenates were stored at –70°C until required.
Western blotting
Samples were denatured in reducing treatment buffer and loaded (200 µg/lane) onto 10% (v/v) Novex Bis-Tris gels (Invitrogen, Paisley, U.K.). After electrophoresis, the proteins were transferred onto nitrocellulose membrane (Bio-Rad) using a Novex blotting apparatus (Invitrogen, Paisley, U.K.) and detected using goat anti-mouse MMP3 (1/250) (R&D, Abingdon, U.K.), HRP-conjugated rabbit anti-goat secondary Ab (DAKO, Cambridge, U.K.), and an ECL Plus kit (Amersham Biosciences, Little Chalfont, U.K.) according to the manufacturer’s instructions. The Ab binding was imaged on ECL film (Amersham Biosciences). For control of protein loading, the blot was stripped with Tris-HCl buffer (62.5 mM, pH 6.7) containing 10 mM 2-ME for 30 min at 60°C with gentle shaking. The membrane was then washed with PBS-Tween 20 for 10 min for 3 times at RT. After blocking with 5% skimmed milk in PBS-Tween 20, the membrane was re-probed with 1/10,000 rabbit anti-mouse -actin (AbCam, Cambridge, U.K.) and 1/1000 HRP-conjugated goat anti-rabbit secondary Ab (DAKO, Cambridge, U.K.). The blot was then developed as described as above.
RNA extraction and quantitative RT-PCR
Total cellular RNA was isolated from frozen colonic tissues by homogenization of the tissue in TRIzol (Invitrogen, Paisley, U.K.) followed by chloroform extraction and isopropanol precipitation. Total RNA was measured at 260 nm by spectrophotometric analysis (Beckman Coulter, High Wycombe, U.K.). Cytokine encoding plasmid (pCMQ2), kindly provided by M. F. Kagnoff (Department of Medicine, University of California, San Diego, CA), was used for quantitative competitive PCR for TNF- transcripts as described in Ref. 13 . To quantify mouse MMP mRNA levels, we constructed a plasmid that encoded a standard RNA using an approach similar to that described in Ref. 13 . Sequences of the oligonucleotide primers used for PCR amplification, and the sizes of the predicted PCR products from the target and standard RNAs are given in Table I. All primers were synthesized and HPLC purified by Sigma Genosys (Sigma-Aldrich). Primers were designed using Primer 3 (Whitehead Institute for Biomedical Research, Cambridge, MA) from cDNA sequences available in PubMed (National Center for Biotechnology Information). The primers span exon-intron boundaries and do not amplify genomic DNA. Sequences are specific for mouse MMP using blast search (National Center for Biotechnology Information), and PCR products were checked and sequenced to verify that the fragment was MMP. The DNA insert containing different MMP primer sites was cloned into pSP64 poly(A) vector (Promega, Southampton, U.K.) to generate poly(A)+ transcripts in vitro. To generate RNA, plasmids were linearized with NotI for pCMQ-2 or EcoRI for mouse MMP plasmid and transcribed in vitro with either T7 or SP6 RNA polymerase under conditions recommended by the supplier (Promega). Serial 10-fold dilutions of standard RNA (106–103 molecules) were co-reverse transcribed with total cellular RNA (1 µg) at 37°C for 50 min in a 20-µl reaction volume containing 50 mM Tris (pH 8.3), 75 mM KCl, 3 mM MgCl2, 3 mM DTT, 10 mM dNTP mix, and 0.5 µg of oligodeoxythymidylate (Amersham Biosciences), using 100 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen). PCR amplification was routinely conducted in 50-µl reaction volume (10 mM Tris (pH 9), 50 mM KCl, 1.5 mM MgCl2, 200 µM each dNTP, and 20 pmol of specific 5'- and 3'-primers), using 1 U of Taq polymerase (Amersham Biosciences). The temperature profile of the amplification consisted of 35 cycles of 45 s denaturation at 94°C, 75 s annealing at 56°C, and 75 s extension at 72°C. PCR products were then separated on a 1% agarose gel (Invitrogen), and the image was taken by a UVP gel documentation system (Fisher Scientific). The band intensities were quantified by Quantity One densitometry software (Bio-Rad). The above protocol allows quantification to 103 cytokine mRNA transcripts per µg of total RNA.
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Mesenteric lymph nodes (MLNs) were removed from infected B10R111 or MMP3–/– mice 5 days postinfection and were washed in calcium-magnesium-free HBSS (Invitrogen). MLNs were then digested with 90 U/ml collagenase 1 and 2.5 U/ml dispase (Sigma-Aldrich) for 1 h at 37°C. The resulting cell suspension was passed through a 30-µm pore size cell strainer and washed in RPMI. DC were stained by FITC-conjugated B220, allophycocyanin-conjugated CD11c, and PE-conjugated CD8 (Caltag Laboratories, Towcester, U.K.) in PBS with 1% BSA for 30 min at 4°C. Corresponding fluorochrome-conjugated isotype controls were also used as negative controls as described in the manufacturer’s instructions. After 3 washings in PBS with 1% BSA at 4°C for 10 min, cells were fixed in 1% paraformaldehyde in PBS. DC were defined as CD11c+B220– cells and were further divided into CD8+ and CD8– populations. DC were then analyzed by FACSCalibur (BD Biosciences, Oxford, U.K.), and the results were analyzed by WinMDI 2.8 software (Scripps Research Institute, La Jolla, CA). The absolute numbers of DCs in the MLNs were calculated from the total number of collected mesenteric lymph node cells (MLNCs) and the proportion of CD11c+B220– cells among them.
Adoptive transfer of CFSE-labeled cells
MLNCs were removed from infected MMP3–/– mice or B10R111 mice 7 days postinfection and were passed through a nylon sieve to release lymphocytes. Cells were washed in RPMI 1640 containing 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich). The lymphocytes were labeled with 10 µM CSFE for 30 min at 37°C (Molecular Probes, Cambridge, U.K.). Labeling was stopped by adding a 5% final concentration of FCS, and the cells were immediately centrifuged and washed with ice-cold PBS. The CFSE-labeled cells from infected MMP3–/– or B10R111 mice (3 x 107) were injected into naive B10R111 or MMP3–/– recipient mice via tail vein (three recipients/group). One day after the cell transfer, mesenteric lymph nodes and colon were removed from B10R111 or MMP3–/– recipient mice, and lymphocytes were prepared as described in Ref. 14 and stained with allophycocyanin-conjugated anti-CD4 mAb for MLNC or PE-conjugated anti-CD3 mAb (Caltag Laboratories). The lymphocytes were then analyzed by FACSCalibur (BD Biosciences) as described above. The absolute numbers of CFSE+CD3+ or CFSE+CD4+ T cells in the CD3+ or CD4+ T cells were calculated from the total number of collected cells and the proportion of CFSE+CD3+ or CFSE+CD4+ T cells among them.
Statistical analysis
The significance of differences between means was determined using the paired t test and the Mann-Whitney U test. p < 0.05 was considered significant.
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Results |
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–/– mice is associated with increased MMP3 transcripts
Mice infected with C. rodentium develop colitis and mount a highly polarized intestinal Th1 response. A component of the response in colonic tissue of infected mice includes increased transcripts for IL-12, IFN-, and TNF-. To determine whether type I cytokines are importance in host defense and mucosal pathology, mice with targeted mutations in the IL-12p40 or IFN- or TNFRp55 gene were orally infected with C. rodentium strain DBS255 (pCVD438) (5, 6). These animals showed various degree of impairment in their ability to clear infection (5, 6). Remarkably, however, mucosal pathology was greater in these mice than in WT controls. The enhanced pathology included increase in crypt length and CD4 cell infiltrates. Therefore, we postulated the enhanced pathology in these cytokine KO mice is due to increased mucosal remodeling. Competitive PCR showed that MMP3 transcripts were significantly greater in infected colonic tissues of cytokine KO mice than infected WT controls (Fig. 1). In addition, MMP3 transcripts were higher in IL-12p40 or IFN- KO mice, which showed more crypt hyperplasia and enhanced pathology than did TNFRp55 KO mice.
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To determine whether MMP3 is important in mucosal thickening, MMP3–/– mice were orally infected with C. rodentium. Infection of WT mice showed an increase in MMP3 protein in the mucosa, and as expected no MMP3 protein was seen in MMP3–/– mice (Fig. 2). Oral infection with C. rodentium caused a substantial increase in crypt hyperplasia in WT mice (Fig. 3, top). However, compared with infected WT controls, MMP3–/– mice showed a similar crypt hyperplasia at various times postinfection (Fig. 3, bottom).
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transcripts in infected colon of MMP3–/– miceInfection with C. rodentium results in heavy infiltrations of CD4+ cells into the colon (Fig. 6). However, in MMP3–/– mice, the increase in CD4+ cell infiltrates in colonic lamina propria was significantly delayed as compared with WT controls (Fig. 6). In WT mice, the numbers of CD4+ cells increased steadily from day 7, peaked on day 21, and declined on day 35 postinfection. However, the numbers of CD4+ cells were low (<300 cells/mm2) in MMP3–/– mice as compared with WT controls (>700 cells/mm2) on day 14. In MMP3–/– mice, the numbers of CD4+ cells increased from day 14 and peaked on day 21.
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transcripts were abundant in colonic tissues of infected WT and MMP3–/– mice (Fig. 7). However, the kinetics of the increase in TNF- transcripts was different between infected WT and MMP3–/– mice, with a delayed increase in the MMP3–/– mice.
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We also conducted a detailed analysis of the kinetics of various MMPs in WT and MMP3–/– mice throughout C. rodentium infection. The number of MMP3 transcripts was low in uninfected colon of WT controls (Fig. 8A). MMP3 transcripts increased rapidly (>4-fold) from day 7, peaked on day 14, and declined from day 21 to a very low number on day 35. As expected, there were no MMP3 transcripts in uninfected and infected colonic tissues of MMP3–/– mice.
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The activities of MMPs are also regulated by endogenous inhibitors TIMPs. TIMP1 transcripts were abundant in colonic tissues infected with C. rodentium (Fig. 8D). The numbers of TIMP1 transcripts in infected colonic tissues increased significantly from day 7, peaked on day 14, and declined to a very low level on days 21 and 35. In addition, the number of TIMP1 transcripts increased significantly (>6-fold) in MMP3–/– mice compared with WT controls.
DCs and CD4 cell migration into the colon
Previous studies have shown that MMP3 is needed for the migration of Langerhans cells from the skin to the draining lymph nodes to initiate cutaneous sensitivity (15). We therefore reasoned that the delayed CD4+ cell response in MMP3–/– mice infected with C. rodentium might have a similar basis. In both WT and MMP3–/– mice, there was a modest increase in B220–CD11c+ cells in the MLNs 5 days after infection. There was no significant difference in DC number between infected WT and MMP3–/– mice (Fig. 9).
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7 integrin expression in uninfected and infected mice (representative images shown in Fig. 11). Only a few 7+ cells were seen in uninfected colon from WT and MMP3–/– mice. In contrast, on day 14 of infection, the colon of WT mice was infiltrated with 7+ cells. In MMP3–/– mice, however, there was also an increase but much less so than in WT mice, consistent with the reduced infiltrate of CD4 cells. MAdCAM was also expressed on vessels of uninfected WT and MMP3–/– mice. On day 14 of infection, there was massive mucosal thickening and the striking appearance of long MAdCAM+ vessels in both groups of mice.
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Discussion |
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expression in the colon. The reduced migration of CD4+ T cells from the donor WT or MMP3–/– mice into the lamina propria of recipient MMP3–/– mice strongly suggests that MMP3 is important in the tissue environment for migration of CD4+ T lymphocytes into the lamina propria.
The nature of the protective immune response needed for sterilizing immunity to this pathogen is still unclear although immunity requires T and B cells (16). We and others have previously shown that mice deficient in B cells or CD4 cells are highly susceptible to infection (17, 18). However, we were unable to clear bacterial infection from the intestine of B cell-deficient mice with immune sera, but recently Bry and Brenner (17) achieved immunity by transferring immune sera into infected CD4-deficient mice. Taken together, these results clearly indicated that immunity needs T cell-dependent Ab production. Nonetheless, we have also shown that cytokine-deficient mice are more susceptible to Citrobacter infection (5, 6). IL-12–/– mice, for example, have very delayed clearance of the bacteria and severe pathology; some mice die; however, serum Ab responses are the same as those in WT mice. Likewise, in this study we also could see no difference in Ab responses between WT and MMP3–/– mice. In the context of this study, our observation that MMP3–/– mice had delayed appearance of CD4+ T cells into the mucosa and reduced TNF- transcripts again suggests a role of CD4+ T cells, perhaps not inclearing the bacteria from the colon but in controlling bacterial number early in the infection.
The balance between MMPs and their inhibitors has been implicated in the tissue remodeling and ulceration in the intestines. In the intestine, stromelysin-1 (MMP3) has been shown to expressed at high levels at the ulcerated areas of clinical IBD and affected areas of celiac diseases (9, 11). In fetal intestinal explant model, MMP3 is the most critical molecule in tissue injury after T cell activation by PWM (19). Furthermore, in animal models, MMP3 is also expressed at high level in colitic samples of the SCID transfer model of colitis and transgenic mice defective in TGF- signaling (20). Also in our study, elevated expression of MMP3 transcripts in colonic tissues is strongly associated with enhanced pathology in infected TNFRp55–/–, IL-12p40–/–, and IFN-–/– mice. However, crypt hyperplasia is induced in infected MMP3–/– mice to an extent similar to that in infected WT mice. The occurrence of crypt hyperplasia in the absence of MMP3 strongly suggests the redundancy of MMPs in tissue remodeling in vivo. The crypt hyperplasia in infected MMP3–/– mice is probably due to the compensatory increase of matrilysin (MMP7) and macrophage metalloelastase (MMP12) transcripts in the infected colon. Due to the similar and broad substrate specificity of different MMPs, matrix degradation could be conducted by other MMPs in tissues where they coexpress and coexert together. Specifically, MMP7 is strongly implicated in bacterial infections and cancers. It is highly expressed in epithelial cells and macrophages and can degrade proteoglycans, gelatins, and elastin (21). MMP12 is macrophage specific and preferentially degrades elastin as well as other matrix components. It is produced predominantly by infiltrating macrophages and appears essential for macrophage migration through the extracellular matrix. In disease, MMP12 has been strongly implicated in pathogenesis in emphysema (22). In addition, the elevated level of TIMP-1 expression on day 14 in infected MMP3–/– mice suggests the homeostatic mechanism to control the excessive activity of MMPs in matrix remodeling. Therefore, it would be interesting to study the phenotypes of MMP7–/– and MMP12–/– mice in C. rodentium infection.
The deficiency in controlling the bacteria in the colon of infected MMP3–/– mice suggests that MMP3 is important in bacteria defense. MMPs are strongly induced in epithelial cells during bacterial infections. MMP7 is strongly induced in epithelial cells of inflamed mucosa of the stomach in Helicobacter pylori infection (23). MMP9 is strongly induced in human gingival epithelial cells infected with Porphyromonas gingivalis (24). MMP7 has been shown to activate antimicrobial peptides, -defensins (cryptdins), in Paneth cells of mouse small intestine (25). MMP7–/– mice are more susceptible to infection with Salmonella typhimurium. The role of MMP3 in bacteria defense is emphasized in that proforms of MMP7 and MMP9 could be proteolytically activated by MMP3.
The reduction of CD4+ lymphocytes in the lamina propria in infected MMP3–/– mice suggests that MMP3 is important in migration of CD4+ lymphocytes into the lamina propria. There are two possibilities by which MMP3 affect the CD4+ T lymphocyte response. MMP3 is required for the migration of Langerhans cells from the skin to the draining lymph nodes to initiate cutaneous sensitivity (15). The first possibility is therefore that mobilization of DCs is impaired in infected MMP3–/– mice, but the similar increase in the number of DCs recovered from draining lymph nodes of infected WT and MMP3–/– mice suggests that there are no intrinsic defects of MMP3–/– mice to mobilize DCs from the intestine into draining lymph nodes. The second possibility of the immune defect of MMP3–/– mice is the inability of CD4+ lymphocytes to home to the intestine. It is evident in the CSFE cell transfer experiment there is a reduced migration of T lymphocytes taken from both WT and MMP3–/– mice into the mesenteric lymph nodes and lamina propria of MMP3–/– mice. Our data therefore suggest that the tissue environment is important in migration of T lymphocytes. Endothelial cells can make MMP3 (26, 27), and there is a possibility that when an intestine-homing CD4+ cell binds to a MAdCAM+ vessel in the colon, it signals to the endothelial cells to release MMP3 into the pericellular space to open up tight junctions, and the lymphocytes migrate through. To support this notion, it has been shown that treatment of mice with MMP inhibitors results in an accumulation of lymphocytes on the lymph node endothelium and reduced diapedesis (28). Whether the MMP inhibitors were acting via lymphocyte-derived MMPs or endothelial-derived MMPs was not established. We also emphasize that in the experiments shown here, recipient animals were not infected with C. rodentium and that endothelial changes induced by infection might add a further layer of complexity to the system. In this regard, it was striking that although there were fewer CD4 cells in the colonic lamina propria of infected MMP3–/– mice on day 14, on days 21 and 35, the numbers were the same. This would suggest that inflammation overrides this early block in lymphocyte extravasation.
Another explanation for the failure of cells to migrate into the intestine could be the absence of the intestine-homing integrin 47 on lymphocytes or endothelial MAdCAM expression in MMP3–/– mice. MAdCAM was expressed basally in MMP3–/– mice, and long, positively staining vessels were seen in the hyperplastic colonic mucosa of infected MMP3–/– mice, making the latter possibility unlikely. Cells infiltrating the colon of infected WT and MMP3–/– mice were also 7 integrin+.
The mechanisms by which the MMP controls T cell migration and bacterial defense remain virtually unexplored. The data presented in this study, using C. rodentium as a model system, add to our understanding of the implications of MMPs in tissue remodeling in the intestine as well as mucosal immunity in general. The identification of other factors in controlling tissue remodeling in vivo and control of T cell migration by MMP are the focus of our current studies.
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Acknowledgments |
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Footnotes |
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1 This work was supported by the Biotechnology and Biological Sciences Research Council. 
2 Address correspondence and reprint requests to Prof. Thomas MacDonald, Mailpoint 813, Division of Infection, Inflammation and Repair, Southampton General Hospital, Southampton, SO16 6YD, U.K. E-mail address: t.t.macdonald{at}soton.ac.uk
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; DC, dendritic cell; MLN, mesenteric lymph node; MLNC, mesenteric lymph node cell; KO, knockout.
Received for publication February 5, 2004. Accepted for publication July 21, 2004.
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References |
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in controlling the number of luminal pathogenic bacteria and immunopathology in infectious colitis. Infect. Immun. 69:6651.. J. Immunol. 168:1804. signalling in the intestine contributes to tissue injury in inflammatory bowel disease. Gut 49:190.v6-mediated TGF- activation causes MMP12-dependent emphysema. Nature 422:169.[Medline]
-defensin activation by the metalloproteinase matrilysin in innate host defense. Science 286:113.This article has been cited by other articles:
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  I. Vanlaere and C. Libert Matrix Metalloproteinases as Drug Targets in Infections Caused by Gram-Negative Bacteria and in Septic Shock Clin. Microbiol. Rev., April 1, 2009; 22(2): 224 - 239. [Abstract] [Full Text] [PDF]
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 L. Bry, M. Brigl, and M. B. Brenner CD4+-T-Cell Effector Functions and Costimulatory Requirements Essential for Surviving Mucosal Infection with Citrobacter rodentium Infect. Immun., January 1, 2006; 74(1): 673 - 681. [Abstract] [Full Text] [PDF]
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 S L-F Pender, V Chance, C V Whiting, M Buckley, M Edwards, R Pettipher, and T T MacDonald Systemic administration of the chemokine macrophage inflammatory protein 1{alpha} exacerbates inflammatory bowel disease in a mouse model Gut, August 1, 2005; 54(8): 1114 - 1120. [Abstract] [Full Text] [PDF]
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