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Published ahead of print on December 4, 2003, doi:10.1165/rcmb.2003-0370OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 761-770, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0370OC

Matrix Metalloproteinase-9–Deficient Dendritic Cells Have Impaired Migration through Tracheal Epithelial Tight Junctions

Hidenori Ichiyasu, Joanne M. McCormack, Karin M. McCarthy, David Dombkowski, Frederic I. Preffer and Eveline E. Schneeberger

Molecular Pathology Unit, Department of Pathology, Massachusetts General Hospital, Charlestown, Massachusetts

Address correspondence to: Eveline E. Schneeberger, M.D., Molecular Pathology Unit, Room 7147, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. E-mail: eschneeberger{at}partners.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
When sampling inhaled antigens, dendritic cells (DC) must penetrate the tight junction (TJ) barrier while maintaining the TJ seal. In matrix metalloproteinase (MMP)-9–deficient mice, in vivo experiments suggest that migration of DC into air spaces is impaired. To examine the underlying mechanisms, we established a well-defined in vitro model using mouse tracheal epithelial cells and mouse bone marrow DC (BMDC). Transmigration was elicited with either macrophage inflammatory protein (MIP)-1{alpha} or MIP-3ß in a time-dependent manner. Control MMP-9+/+ BMDC cultured with granulocyte macrophage–colony-stimulating factor for 7 d showed a 30-fold greater transepithelial migration toward MIP-3ß than MIP-1{alpha}, indicating a more mature DC phenotype. MMP-9–/– BMDC as well as MMP-9+/+ BMDC in the presence of the MMP inhibitor GM6001, although showing a similar preference for MIP-3ß, were markedly impaired in their ability to traverse the epithelium. Expression levels of CCR5 and CCR7, however, were similar in both MMP-9–/– and MMP-9+/+ BMDC. Expression of the integral TJ proteins, occludin and claudin-1, were examined in BMDC before and after transepithelial migration. Interestingly, occludin but not claudin-1 was degraded following transepithelial migration in both MMP-9–/– and control BMDC. In addition, there was a > 2-fold increase in claudin-1 expression in MMP-9–/– as compared with control BMDC. These observations indicate that occludin and claudin-1 are differentially regulated and suggest that the lack of MMP-9 may affect claudin-1 turnover.

Abbreviations: bronchoalveolar lavage, BAL • bone marrow–derived dendritic cells, BMDC • bovine serum albumin, BSA • confocal laser scanning microscopy, CLSM • dendritic cells, DC • extracellular matrix, ECM • epidermal growth factor, EGF • fetal calf serum, FCS • fluorescein isothiocyanate, FITC • granulocyte macrophage–colony-stimulating factor, GM-CSF • intercellular adhesion molecule, ICAM • interleukin, IL • macrophage inflammatory protein, MIP • matrix metalloproteinase, MMP • ovalbumin, OVA • phosphate-buffered saline, PBS • polymerase chain reaction, PCR • phycoeryhtrin, PE • reverse transcriptase, RT • transepithelial electrical resistance, TER • tissue inhibitor of metalloproteinase, TIMP • tight junction, TJ


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A number of physical and functional barriers serve to protect the lung from the harmful effects of inhaled environmental factors to which it is exposed (1). Should these barriers be breached, however, the immune system must distinguish innocuous from pathogenic agents, while at the same time limiting tissue damage resulting from an immune response. Failure to do so can result in asthma, idiopathic fibrosis, or granulomatous inflammation. The key role of the dendritic cell (DC) in the immune surveillance of the lung is incontrovertible (2–5). Following an inhaled antigen challenge, DC precursors are rapidly recruited into the lung interstitium (6), airway epithelium (7), and into alveolar spaces (8). Conversely, when antigen primed DC are administered intratracheally, they enter the lung and migrate to thoracic lymph nodes to initiate a T cell response (9). During all this DC trafficking, there must be mechanisms in place that maintain the integrity of the lung epithelial barrier. In the intestine, this problem appears to have been solved by DC forming transient tight junctions (TJ) with epithelial cells as they intercalate between them and extend dendritic processes into the intestinal lumen to sample the microbial flora (10). Whether a similar mechanism pertains to the lung is not known.

After antigen acquisition, DCs undergo phenotypic changes as they migrate via lymphatics to local lymph nodes. To accomplish this requires not only that DC follow chemoattractant gradients, but that they produce enzymes that enable them to traverse the extracellular matrix (ECM) and that activate latent cytokines involved in the immune response (11). Although some leukocytes are known to secrete matrix metalloproteinases (MMP) (12) and collagenase (13), their role in neutrophil migration has been questioned (14). It is only recently that a role for MMPs in DC biology has been examined. In dinitrofluorobenzene-induced contact hypersensitivity, T cells from MMP-3–/– mice had a markedly impaired contact hypersensitivity response. Based on data from in vitro studies (15), it is suggested that without MMP-3 to degrade E-cadherin, Langerhans cells (LC) were unable to emigrate from the epidermis to local lymph nodes. In contrast, a prolonged contact hypersensitivity response was noted in MMP-9–/– mice. In the absence of MMP-9 LC, keratinocytes and/or macrophages failed to produce active, anti-inflammatory interleukin (IL)-10, a response that was upregulated in wild-type controls (16). Emigration of LC and dermal DC from skin explants was reduced by the injection of both anti–MMP-2 and anti–MMP-9 antibodies, as well as in explants from MMP-9–/– mice (17). In an experimental model of asthma, peribronchial mononuclear cell infiltration was impaired in MMP-9–/– mice (18). Following antigen challenge of mice, MMP-12 and MMP-14 mRNA was increased in wild-type, but not in MMP-9–/– mice. By contrast, in IL-13 transgenic, MMP-9–/– or IL-13 transgenic, MMP-12–/– mice, induced to express IL-13, the absence of MMP-12, but not MMP-9, reduced the number of eosinophils and macrophages recovered by alveolar lavage. Interestingly, IL-13 induced the expression of MMP-2, -12, -13, and -14 in lung lysates of MMP-9–/– mice, but reduced the expression of MMP-2, -9, -13, and -14 in MMP-12–/– mice (19). In MMP-2–/– mice, complete aspergillus antigen (CAA)-induced asthma resulted in large numbers of inflammatory cells accumulating in the peribronchial interstitium with markedly impaired migration of these cells into the airspaces of lung (20). None of these studies focused on lung DC.

Regulation of some MMP activity requires the interaction of several proteins. For example, CD44, expressed abundantly on DC (21, 22), is a docking site for activated MMP-9 (23). The enzyme would thus be strategically placed to cleave components of the interstitium as DC migrate through interstitial compartment. Another example is the activation of pro–MMP-2, which requires the interaction of soluble tissue inhibitor of metalloproteinase (TIMP)-2 and MMP-14 on the cell surface (11).

In the present study we show that the absence of MMP-9 in bone marrow DCs (BMDCs) from MMP-9–/– mice markedly impairs their migration in response to a chemotactic signal. This is not due to a difference in chemokine receptor expression, because BMDC from both MMP-9+/+ and MMP–/– mice express similar amounts of CCR5 and CCR7. We further show that BMDCs from both MMP-9+/+ and MMP-9–/– mice maintain transepithelial electrical resistance (TER) by forming what appear to be transient, functional TJs with airway epithelial cells. Although BMDC from both control and MMP-9–deficient mice express occludin and claudin-1 at both mRNA and protein levels, we report the novel observation that claudin-1 expression is significantly upregulated in MMP-9–deficient DC. Furthermore, following migration through epithelial TJs, intact claudin-1 continues to be expressed, but occludin is degraded in both control and MMP-9–/– DC, suggesting that these two integral TJ proteins are differentially regulated in BMDC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Antibodies
Cell culture media included RPMI 1640 (Cellgro Mediatech, Herndon, VA) and Ham's F-12 (GIBCO, Grand Island, NY). Bovine serum albumin (BSA), ovalbumin (OVA), gentamicin, 2-mercaptoethanol(2-ME), transferrin, insulin, 3,3',5-triiodo-L-thyronine, hydrocortisone, and type 1 rat tail collagen were obtained from Sigma Chemical Co. (St. Louis, MO). The mouse granulocyte macrophage–colony-stimulating factor (GM-CSF) hybridoma was a gift from the former Genetics Institute (Cambridge, MA). Fetal calf serum (FCS), epidermal growth factor (EGF), and endothelial cell growth supplement (ECGS) were from BD Biosciences (Bedford, MA). Recombinant mouse macrophage inflammatory protein (MIP)-1{alpha} and MIP-3ß were purchased from R&D Systems (Minneapolis, MN). Monoclonal antibodies (mAb) were generated from the following hybridomas: M5/114 (anti-MHC class II, TIB 120), GK1.5 (anti-CD4, TIB 207), HO2.2 (anti-CD8, TIB 150), B21–2 (anti-Iab,d, TIB 229), RA3–3A1/6.1 (anti-B cell, TIB 146) (American Type Culture Collection, Rockville, MD), and RB6 (anti-granulocyte; DNAX, Palo Alto, CA). Peroxidase-conjugated goat anti-rabbit IgG and rabbit anti-mouse IgG (Sigma), rabbit anti-occludin, rabbit anti–claudin-1, and mouse anti–claudin-4 (Zymed, South San Francisco, CA) were purchased as indicated. Phycoerythrin (PE)-conjugated rat anti-mouse CCR5 mAb (C34–3448), hamster anti-mouse CD11c (HL3), allophycocyanin (APC)-conjugated rat anti-mouse CD11b (M1/70), fluorescein isothiocyantate (FITC)-conjugated rat anti-mouse CD45R/B220 (RA3–6B2), biotin-conjugated rat anti-mouse I-A/I-E (2G9), streptavidin perdinin chlorophyll-a protein (Sav-PerCP), and rat anti-mouse Fc{gamma} II/III receptor (CD16/CD32) mAb (2.4G2) were from BD Pharmingen (San Diego, CA). Alexa 488–conjugated goat anti-rabbit IgG, CY3-conjugated goat anti-rat IgG, and streptavidin-PE were from Molecular Probes (Eugene, OR). Biotinylated goat anti-human IgG was from Caltag Laboratories (Burlingame, CA). CCL19-Ig chimeric protein was a generous gift of U. H. von Andrian (Center for Blood Research, Boston, MA) (24). Rabbit complement (Accurate Chemical and Science, Westbury, NY), Nutridoma (Boehringer Mannheim, Indianapolis, IN), chloral hydrate, paraformaldehyde (Fisher Scientific, Fair Lawn, NJ) were purchased as indicated. The hydroxamic acid dipeptide analog inhibitor of MMPs, N-[(2R)-2-(Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-trp-methylamide (GM6001), and the inactive peptide control, N-t-Butoxycarbonyl-L-leucyl-L-trp-methylamide were obtained from Calbiochem (San Diego, CA) and dissolved in DMSO as a 25 mM solution and stored at –20°C.

Animals
MMP-9–deficient mice on an FVB background were generated by targeted mutagenesis as described previously (25), and were obtained as a generous gift from Z. Werb, UCSF, San Francisco, CA. They and their wild-type littermates were housed in a pathogen-free animal facility at the Massachusetts General Hospital. Homozygocity was confirmed by periodic genotyping. Mice aged 8–12 wk were used in all experiments. All experiments were conducted in accordance with Massachusetts General Hospital and National Institutes of Health guidelines for care and use of laboratory animals.

Epithelial Cell Culture
The mouse tracheal epithelial cell line (MTE 7b) was a generous gift from R. C. Boucher, UNC, Chapel Hill, NC (26). They were cultured at 33°C in 5% CO2, 95% room air in 24-mm Transwell-COL culture inserts (Corning-Costar, Cambridge, MA). Culture medium consisted of a 1:1 mixture of Ham's F-12 and 3T3 fibroblast-conditioned medium supplemented with 2.5 µg/ml transferrin, 5 µg/ml insulin, 12.5 µg/ml EGF, 1.875 µg/ml ECGS, 15 nM tri-iodothyronine, 0.5 µM hydrocortisone, and 0.5 mM CaCl2. For chemotaxis experiments, cells were plated at a density of 2 x 105 cells/cm2 on polycarbonate membranes (3.0 µm pore size, 12 mm diameter) of Transwell cell culture inserts coated with type 1 rat tail collagen (0.4 mg/ml), for AP to BL transmigration. Monolayers were also cultured on the lower surface of the insert as described previously (27), with slight modification. Briefly, sterile polypropylene collars with an inner diameter equal to the outer diameter of the inserts and a height of 10 mm were tightly fixed to the inverted inserts. MTE 7b cell suspensions were added to the inverted inserts and cultured for 16 h. The collars were then removed and the inserts were placed upright in 12-well culture plates with fresh culture medium, for BL to AP transmigration. The integrity of MTE cell monolayers was monitored by daily TER measurements, using a Millicell-ERS epithelial volt-ohmmeter (World Precision Instruments, New Haven, CT) under temperature-controlled conditions. TER ({Omega} x cm2) was calculated by subtracting the contribution of the bare membrane and medium and multiplying by the surface area of the membrane. Experiments were conducted 3–4 d after plating when TER was > 150 {Omega} x cm2. Monolayers were given a fresh medium change 24 h before the transmigration assay.

BMDC Preparation
BMDCs were generated as described (28), with some modifications. Control and MMP-9–/– mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.4 mg/g of body weight). Bone marrow was obtained from femurs and tibias of these mice, and passed through a cell strainer (70 µm pore size; Becton Dickinson, Sunnyvale, CA). Red blood cells were lysed in aqueous ammonium chloride (150 mM NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.3). To deplete MHC class II+ cells, T and B lymphocytes, they were incubated in a mAb cocktail consisting of GK1.5, HO2.2, B21–2, and RA3–3A1/6.1 hybridoma supernatant for 1 h at 4°C, then treated with rabbit complement for 1 h at 37°C. The complement-depleted BM cells were resuspended in complete medium (RPMI 1640, 5% FCS, 50 µM 2-ME, 20 µg/ml gentamicin, and 1,000 U/ml GM-CSF), and plated at 1 x 106/ml in 24-well plates (Becton Dickinson, Franklin Lakes, NJ). Cultures were fed every 2 d by aspirating 50% of the medium, and replacing with an equal volume of fresh complete medium. At Day 6, cell aggregates and floating cells were harvested and contaminating neutrophils were eliminated by complement lysis using RB6 mAb. Cells were plated at 1 x 106/ml in a 100-mm tissue culture dish (Corning-Costar) in complete medium for 1 h. Nonadherent cells were carefully washed away and adherent cells were cultured overnight in complete medium. The next day the nonadherent cells were harvested and replated again. After a 2-h incubation, the nonadherent cells were collected as BMDC, at > 90% pure and > 90% viable, as determined by immunostaining and trypan blue dye exclusion, respectively.

DC Transepithelial Migration Assay
To eliminate MMP activity present in serum, all transmigration studies were conducted in serum-free medium (RPMI 1640, 1% Nutridoma, 50 µM 2-ME, 20 µg/ml gentamicin, and 1,000 U/ml GM-CSF). Before each assay, confluent epithelial cell monolayers were washed twice with prewarmed medium and transferred to a new 12-well plate. For AP to BL transmigration, 4 x 105 BMDC in 0.5 ml of prewarmed medium was added to the insert and 1.5 ml of chemokine containing medium was added to the well. For BL to AP transmigration, 1.5 ml of chemokine containing medium was first added to the well and 4 x 105 BMDC in 0.5 ml of prewarmed medium was added to the insert. The Transwells were incubated at 33°C in a humidified tissue culture incubator. At timed intervals, the cells that had migrated into the lower compartment were harvested, re-suspended in 200 µl of phosphate-buffered saline (PBS) and counted in a hemocytometer. Integrity of the monolayer was assessed by TER measurements. Monolayers were then processed for confocal laser scanning microscopy (CLSM) by staining them for occludin and the BMDC for MHC class II antigen (see below).

CLSM
Expression of TJ proteins and localization of BMDC in the monolayers were assessed by CLSM. Briefly, the monolayers were fixed in 3.7% paraformaldehyde in PBS for 10 min at RT, and permeabilized with 0.2% TX-100 in PBS for 10 min at RT. Tight junctions were labeled with rabbit anti-occludin (4 µg/ml) pAb followed by Alexa 488–conjugated goat anti-rabbit IgG. BMDC were immunolabeled with M5/114 mAb (anti-MHC class II) followed by CY3-conjugated goat anti-rat IgG. Coverslips were mounted with Prolong anti-fade (Molecular Probes) and analyzed by CLSM (LSM 510 invert; Carl Zeiss, Thornword, NY) equipped with an argon and a helium-neon laser for excitation at 488 and 543 nm and BP505–530 and LP560 emission filters. Serial sections were collected starting at the apical surface at 0.049 µm pixel size and a step increment of 0.2 µm in the z-axis. Cross-section xz images were rendered using LMS 510 software. Images are presented as a two-color composite in which TJs appear green and BMDC appear red.

Electron Microscopy
MTE 7b monolayers with included BMDC were fixed with paraformaldehyde lysine periodate (PLP) fixative (29) for 10 min at 4°C, rinsed in PBS, quenched with 0.05 M glycine, and blocked with 1% BSA in PBS. The monolayers were then incubated with M5–114 mAb in 1% BSA in PBS for 1 h at RT, rinsed in BSA/PBS, and incubated with goat anti-rat IgG conjugated to 15 nm gold (Ted Pella, Inc., Redding CA) for 90 min at RT. After several rinses in PBS, the monolayers were again fixed with PLP fixative and processed for Epon embedding. Sections were examined in a Philips 301 electron microscope (Einthoven, Holland).

Flow Cytometry
Cells (0.5–1 x 106) were incubated with CD16/CD32 (anti-mouse Fc{gamma} II/III receptor) for 5 min at 4°C in wash buffer (PBS containing 0.1% NaN3 and 1% normal mouse serum) to block Fc receptors. To examine chemokine receptors on BMDC, cells were immunolabeled with PE-conjugated anti-CCR5 for 20 min at 4°C. Mouse CCR7 was detected by incubating 1 x 105 cells with 0.1 ml of CCL19-Ig supernatant for 1 h at 4°C followed by biotinylated goat anti-human IgG and PE-streptavidin as described (24). To analyze DC in bronchoalveolar lavage (BAL), cells were incubated with FITC–anti-CD45R/B220, APC–anti-CD11b, PE–anti-CD11c and biotinylated anti-I-A/I-E, followed by streptavidin-PerCP. All samples were analyzed with an FACS-Vantage flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson). Cells were electronically gated according to light scatter properties to exclude cell debris. Propidium iodide was added in the last wash to exclude dead cells from analysis.

Reverse Transcriptase–Polymerase Chain Reaction
To detect mRNAs for selected MMPs and TJ proteins in BMDC, primers were designed from murine sequences provided by the National Center for Biotechnology Information, Bethesda, MD. These were determined to be unique and not to contain sequences in other known murine TJ protein or MMP cDNAs. The primers were synthesized by MWG-Biotech, High Point, NC (Table 1). Total RNA from purified BMDC and MTE 7b cells was extracted by using RNAqueous-4PCR purification kit (Ambion, Austin, TX). The RNA was cleaned by treatment with RNase-free DNase I (Ambion). Reverse transcriptase (RT)-polymerase chain reaction (PCR) was performed using the ProSTAR HF single-Tube RT-PCR System (Stratagene, La Jolla, CA). Briefly, 100 ng of total RNA and Moloney murine leukemia virus reverse transcriptase were used to reverse transcribe cDNA at 42°C for 15 min. After inactivation of the reverse transcriptase at 95°C for 1 min, the cDNA fragments were amplified by 40 cycles of PCR (denaturing at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 68°C for 2 min) using GeneAmp PCR System 9,700 (PerkinElmer, Norwalk, CT). PCR products were visualized by ethidium bromide agarose gel electrophoresis and ultraviolet transillumination (Fluor-S Multi-Imager; Bio-Rad Laboratories, Philadelphia, PA). A negative control without RT was included in each reaction. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control.


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  		TABLE 1. RT-PCR primers used to detect selected MMP and TJ protein mRNA

 
SDS-PAGE
MTE 7b cell monolayers were rinsed and scraped into ice-cold TBS (10 mM Tris-HCl, 150 mM NaCl, pH 8.0) and centrifuged. Cell pellets of MTE 7b cells or BMDC were lysed in 2% SDS in TBS with inhibitors (Protease inhibitor cocktail tablets, 5 µg/ml DNase I, 10 nM sodium pyrophosphate, 1 mM Na3VO4, 20 mM NaF; Roche Applied Science, Indianapolis, IN). Lysates were boiled for 5 min with intermittent vortexing and sheared six times through a 27-gauge needle. Protein concentration was determined using a Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA) and BSA as a standard. Equal amounts of protein (20 µg/lane) were resolved in 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA) by SDS-PAGE under reducing conditions and transferred electrophoretically to polyvinylidene difluoride membranes (Invitrogen). BMDC from the starting pool and migrated BMDC were rinsed with ice-cold TBS, centrifuged, and lysed as described above.

Western Blotting
Polyvinylidene difluoride membranes were blocked with 5% nonfat dry milk in TBS containing 0.05% Tween-20. They were incubated with rabbit anti-occludin (0.3 µg/ml), rabbit anti–claudin-1 (0.4 µg/ml), or mouse anti–claudin-4 (0.8 µg/ml) for 1 h at room temperature, followed by horseradish peroxidase–conjugated goat anti-rabbit IgG (1:5,000) or rabbit anti-mouse IgG (1:5,000) for 1 h at room temperature. Detection of occludin and claudin-4 was with Western Lightning Chemi-luminescence Reagent Plus (PerkinElmer, Boston, MA). Detection of claudin-1 was with Super Signal West Femto Maximum kit (Pierce Chemical Co., Rockford, IL). Protein bands were quantified by densitometry using a Fluor-S Multi-Imager and Quantity One software (Bio-Rad).

Ovalbumin-Induced Asthma Model
To determine whether our in vitro observations reflect in vivo conditions, MMP-9–/– and control mice were immunized by intraperitoneal injection of 100 µg OVA in 0.2 ml alum (Pierce) or alum alone (control) on Days 0 and 7. On Days 14–21, mice were challenged on eight consecutive days by intranasal administration of 100 µl of 1% ovalbumin (OVA) in PBS or PBS alone (control), under light anesthesia. At 24 h after the last challenge, mice were killed and BAL was performed, using a total 4.8 ml (8 x 0.6 ml) of 0.1 mM EDTA in PBS. Total cell numbers in BAL were counted using a hemocytometer. Cytospin slides of BAL cells were prepared using a cytocentrifuge (Thermo Shandon, Pittsburgh, PA), stained with Diff-Quick solution (Fisher Scientific) and differential cell counts were determined. The remaining cells were analyzed by four-color flow cytometry.

Statistics
Data are presented as the mean ± SD. Statistical analysis was done with InStat software (GraphPad software, San Diego, CA). Values were considered significantly different when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In response to inhaled antigens, DCs traverse basement membranes and the interstitium to reach the mucosal surface of airways and the lung, a process that requires the enzymatic activity of MMPs (11). To sample inhaled antigens at the mucosal surface, DC must penetrate TJs without disrupting the mucosal barrier. To determine whether this might be accomplished by forming transient TJs, we established a well defined in vitro chemotaxis assay in which TER of MTE 7b cell monolayers could be monitored during chemokine induced BMDC transmigration. The contribution of MMP-9 to this process was examined using BMDC harvested from MMP-9–deficient mice and comparing them to those harvested from their normal littermate controls.

mRNA Expression of Selected MMPs and TJ Proteins by BMDC from MMP-9–/– and Control Mice and by MTE 7b Cells
RT-PCR performed on BMDC from control MMP-9+/+ mice revealed expression of MMP-2, -9, and -14 mRNA, whereas those from MMP-9–/– mice expressed MMP-2 and -14, but no MMP-9 mRNA. MMP-2 and -14, but no MMP-9 mRNA, was detected in MTE 7b cells (Figure 1A).



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  		Figure 1. Expression of MMP and TJ protein mRNA in BMDC and MTE cells. Total RNA from MTE cells and from BMDC of MMP-9–/– mice and their littermate controls was extracted. mRNA encoding MMP-2, -9, and -14 and the TJ proteins occludin, claudin-1, -4. -5, and -8 was examined by RT-PCR analysis as described in MATERIALS AND METHODS. (A) BMDC from control mice expressed MMP-2, -9, and -14, whereas those from MMP-9–/– mice expressed MMP-2 and -14 mRNA. MMP-2 and -14 were detected in MTE cells. (B) BMDC from both control and MMP-9–/– mice expressed occludin, claudin-1, and claudin-5 mRNA, whereas occludin, claudin-1, -4, and -8, but not claudin-5, were detected in MTE cells.

 
Interestingly, BMDC isolated from both MMP-9–/– mice and their MMP-9+/+ littermate controls expressed occludin, claudin-1, and claudin-5, but no claudin-2, -4, or -8 mRNA. By contrast, MTE 7b cells expressed occludin, claudin-1, -4, and -8 mRNA (Figure 1B).

Time Course Experiments Using the In Vitro Chemotaxis Assay
To establish optimal conditions, MTE 7b cells were plated in triplicate at 50% confluence in collagen-coated Transwell inserts and TER was monitored daily. After a TER of ~ 150 {Omega} x cm2 was attained, 4 x 105 purified, cultured control BMDC were added to the inserts and 200 µg/ml of either MIP-1{alpha} or MIP-3ß added to the well. Migrated BMDC were harvested from the wells after 2, 4, 8, and 16 h and counted. MIP-1{alpha} did not elicit a strong chemotactic response. In fact only ~ 2% of the starting BMDC population was retrieved in the well at 16 h (Figure 2A). By contrast, ~ 50% of the starting population had migrated into the well in response to MIP-3ß over the same time period (Figure 2B).



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  		Figure 2. Time course of control BMDC transepithelial migration in response to chemokines. BMDC from 7-d cultures were tested in transepithelial migration assays using MTE cell monolayers. BMDC (4 x 105 cells) were added to the apical aspect of epithelial cell monolayers and their migration in response to MIP-1{alpha} or MIP-3ß (200 ng/ml) was assessed at 2, 4, 8, and 16 h by counting the number of BMDC in the lower compartment. BMDC migration in response to (A) MIP-1{alpha} (filled circles) or (B) MIP-3ß (filled circles) occurred in a time-dependent manner. Open circles, control. The response of BMDC to MIP-3ß was 30-fold greater than to MIP-1{alpha}. Data are the mean ± 1 SD of triplicate samples. Similar experiments were conducted in collagen coated Transwell inserts without MTE cell monolayers. In the absence of a cell monolayer, the number of BMDC retrieved from the well was increased severalfold, regardless of whether cytokine had been added to the well or not (16- and 3,000-fold at 4 h and 4- and 29-fold at 16 h, respectively). (C) At the end of the assay the monolayers from each time point were immunolabeled for occludin (green) and for MHC class II antigen (red) in BMDC, and examined by confocal microscopy. Images represent xy sections taken at the level of the TJs and from which xz images were derived. At earlier time points (2–4 h) many of the BMDC were present at the level of epithelial tight junctions. Bar is 80 µm.

 
To examine the interaction of BMDC with MTE cells during the chemotaxis assay, the monolayers were immunolabeled for occludin using an FITC-labeled (green) secondary antibody, and MHC class II on BMDC was detected with CY3-labeled (red) secondary antibody. In a representative MIP-3ß–induced chemotactic assay, the images in Figure 2C reveal that at 2 and 4 h numerous BMDC were in the process of traversing the epithelial cell monolayer. By 16 h, very few BMDC remained on the surface of the MTE cell monolayer, and the number of actively transmigrating BMDC was greatly diminished. The significantly lower response elicited by MIP-1{alpha}, as compared with MIP-3ß, suggested that BMDC cultured for 7 d with 1,000 U/ml of GM-CSF produced relatively mature DC. Based on these timed experiments, most of the chemotaxis assays with MMP-9–/– BMDC were conducted over a 16-h period.

TER Is Maintained during BMDC Transepithelial Migration, but Expression of TJ Proteins Is Differentially Regulated in BMDC
During the chemotaxis assays, the functional integrity of epithelial TJs was monitored by TER measurements. TER measures ion conductivity of the TJ and, therefore, is a stringent measure of its integrity. Although there was some variability in the maximum TER achieved among MTE cell monolayers, TER was maintained during the assay (Figure 3A), suggesting that a functional seal was formed between BMDC and MTE cells during the migration process. The more active transepithelial migration induced by MIP-3ß, as compared with MIP-1{alpha}, was associated with a somewhat lower TER, suggesting that the heterotypic seal formed between migrating BMDC and epithelial cells is less tight than the homotypic seal between epithelial cells. To further evaluate the interaction between DC and epithelial cells, MTE cell monolayers were examined at a higher magnification by CLSM and by electron microscopy. In the xy images shown in Figure 3B, the plane from which the xz images were derived is indicated by a horizontal green line (Figure 3B, left) or by a horizontal red line (Figure 3B, right). The yellow dots seen in Figure 3B indicate an intimate contact between the CY3 labeled BMDC cell membrane (red) and occludin (green) expressed at the TJ of the MTE 7b cell. To definitively identify migrating BMDC by electron microscopy, pre-embedding immunogold labeling for MHC class II was conducted on the BMDC that were migrating through MTE cell monolayers. The ultrastructural preparations revealed a focal close apposition between the membranes of the two cell types with an accompanying cytoplasmic plaque consistent with the appearance of a TJ-like seal (Figure 3C and boxed area).



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  		Figure 3. Chemokine induced transmigration of BMDC across MTE 7b cell monolayers: TER measurements, confocal microscopy, electron microscopy. (A) In representative experiments TER of MTE 7b cell monolayers are maintained above 100 {Omega} x cm2 during MIP-1{alpha}- or MIP-3ß–induced transepithelial chemotaxis (shaded rectangle), in both AP to BL (black and white circles) and BL to AP (black and white triangles) directions, of MMP-9–/– (open symbols) and control BMDC (filled symbols). (B) After a 4- and 16-h chemotaxis assay, MTE cell monolayers were immunolabeled for occludin (green) and for MHC class II antigen on BMDC (red). CLSM images represent xy sections taken at the level of the TJ and reconstituted in xz sections. The black arrow on the right denotes the direction of BMDC transepithelial migration. The open arrow on the left indicates the level of the TJ in MTE cell monolayers where the migrating DCs are located. Bar is 20 µM. (C) At the end of the chemotaxis assay, MTE cell monolayers were fixed, immunogold labeled for MHC class II antigen to identify BMDC, and processed for transmission electron microscopy. A transmigrating, immunogold-labeled (arrowheads) DC is shown between two MTE cells. The boxed area is magnified to show a TJ formed between the MTE cell and the migrating DC. Bar is 700 nm; bar in insert is 250 nm.

 
Occludin Is Degraded following Transepithelial Migration, whereas MMP-9 Deficiency Is Associated with Upregulated Claudin-1 Expression in BMDC
During BMDC migration through MTE cell monolayers, we observed that: (i) TER did not change appreciably; (ii) ultrastructural studies were consistent with TJ-like structures formed between BMDC and the MTE cells; and (iii) BMDC express occludin, claudin-1, and claudin-5 mRNA. We therefore examined BMDC for expression of TJ proteins by Western blotting. Occludin was detected as a 60-kD band in both MMP-9–/– and MMP-9+/+ BMDC before they were added to MTE cell monolayers, as well as in MTE 7b cells (Figure 4). However, when BMDC were retrieved from the well 4 h after migrating through the epithelial cell monolayer, virtually all occludin was degraded to a < 50-kD form in both MMP-9–deficient and control BMDC. Interestingly, the level of claudin-1 expression remained unchanged after transmigration through the epithelial cell monolayer. Moreover, claudin-1 expression was increased > 2-fold in MMP-9–/– as compared with MMP-9+/+ BMDC, both before and after transepithelial migration (Figure 4).



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  		Figure 4. TJ proteins are differentially regulated in migrating BMDC. MTE cell monolayers were plated on collagen-coated Transwell inserts (for AP to BL BMDC migration) and MMP-9+/+ or MMP–/– BMDC were added to the insert as described in MATERIALS AND METHODS. Chemotaxis was initiated by addition of MIP-3ß to the well, and migrated BMDC were harvested from the well 4 h later. The MTE cells were obtained from inserts with no added BMDC. To check for possible contamination, blots were probed for claudin-4 that is expressed in MTE cells, but not in BMDC. Equivalent amounts of occludin were detected in MMP-9+/+ and MMP-9–/– BMDC before chemotaxis; however, after transepithelial migration virtually all the occludin is degraded in both BMDC populations. BMDC from MMP-9–/– mice express approximately twice as much claudin-1, relative to controls. In contrast to occludin, claudin-1 is not degraded following transepitlhelial migration. No clauidin-4 was detected in any of the BMDC samples. Molecular weights are indicated to the right of the figure.

 
Chemical Inhibition of MMP Activity Reduces BMDC Migration across Epithelial Cell Monolayers
To determine whether MMPs facilitate transepithelial migration of BMDC, chemotactic assays across MTE 7b cell monolayers were conducted in the AP to BL direction in the presence of the synthetic broad-spectrum MMP inhibitor GM6001, or with its inactive control peptide, both at 25 µM. The effect of MMP inhibition on either MIP-1{alpha}– or MIP-3ß–induced chemotaxis was tested. There was a > 3- and 7-fold reduction in transepithelial migration of BMDC in response to MIP-1{alpha} and MIP-3ß, respectively, when GM6001 was present (Figures 5A and 5B). A similar reduction was observed in GM6001-treated BMDC induced to transmigrate in a BL to AP direction by MIP-3ß (data not shown). BMDC that had migrated through the MTE cell monolayer in the presence of either GM6001 or inactive control peptide were viable as judged by trypan blue dye exclusion. The integrity of the MTE cell monolayer was maintained throughout the assay as determined by TER measurements and immunofluorescent staining of the monolayer for occludin. Although these results clearly indicate that MMPs facilitate the migration of DC across epithelial cell monolayers, because GM6001 is a broad-spectrum inhibitor of MMPs, they do not clarify which of the many MMPs are specifically involved.



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  		Figure 5. Effect of a broad-spectrum MMP inhibitor (GM6001) or the deletion of MMP-9 on transepithelial migration of BMDC. (A and B) BMDC from control mice were tested in a 16 h, AP to BL chemotaxis assay in the presence of either a broad spectrum MMP inhibitor, GM6001, or an inactive peptide control (both at 25 µM). Both MIP-1{alpha} and MIP-3ß induced transmigration was significantly inhibited by GM6001. Data are the mean ±1 SD of triplicate samples. (C and D) BMDC from control and MMP-9–/– mice were tested in a 16-h AP to BL or BL to AP chemotaxis assay. Both the MIP-1{alpha} and MIP-3ß induced transepithelial migration in either direction was significantly less in MMP-9–/– BMDC than in controls. Of note is that the magnitude of the MIP-3ß–induced migration of both control and MMP-9–/– BMDC is an order of magnitude higher than with MIP-1{alpha}. Data are the mean ± 1 SD of two MIP-1{alpha}– and three MIP-3ß–induced assays, all of which were conducted in triplicate. *P < 0.05, **P < 0.01 as compared with controls. Further control experiments using Transwell inserts without MTE cell monolayers resulted in a 4-fold and 14-fold increase in BMDC from MMP-9+/+ and MMP-9–/– mice, respectively.

 
MMP-9 Deficiency Impairs Transepithelial Migration of BMDC
To examine the contribution of MMP-9 to BMDC transepithelial migration specifically, we compared the chemotactic response of BMDC from MMP-9–/– mice with those of their MMP-9+/+ littermate controls. During the course of isolating BMDCs, we noted a consistently lower yield of total bone marrow cells and BMDC from MMP-9–/– mice as compared with controls (Table 2). This was somewhat surprising because it has been reported that MMP-9–/– mice have a normal number of circulating leukocytes (30). We also noted that relative to BMDC from control mice, those harvested from MMP-9–deficient mice adhered to the tissue culture wells and required more vigorous rinsing to retrieve them. MMP-9 is involved in the release of soluble Kit-ligand (sKL) from endothelial and hematopoietic stem cells (HSC). In MMP-9–/– mice release of sKL and the motility of HSC are impaired, which may account, in part, for our observations (31).


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  		TABLE 2. Yield of total bone marrow cells and BMDC from MMP-9–/– and MMP-9+/+ mice

 
To determine whether the polarity of migration across the MTE cell monolayer was influenced by (i) the chemokine, (ii) the expression of MMP-9, or (iii) the level of maturity of the DC, as judged by their ability to respond to either MIP-1{alpha} (immature DC) or MIP-3ß (mature DC), the following assays were conducted. MTE cell monolayers were cultured either on the top or bottom surface of the collagen-coated insert (Figure 6B), and 4 x 105 BMDC were added to the insert and 200 µg/ml of either cytokine was added to the well. Surprisingly, MIP-1{alpha}–induced migration of control BMDC was significantly less (P < 0.01) (Figure 5C) in the physiologic direction (BL to AP) than in the opposite direction (AP to BL). By contrast, no directional preference was observed in MIP-3ß–induced migration of control BMDC (Figure 5D). As observed in the timed chemotactic experiments, the number of BMDC that migrated in response to MIP-1{alpha} was an order of magnitude less than when MIP-3ß was applied, indicating that these were relatively mature DC. However, irrespective of the chemokine applied and the direction of migration tested, BMDC from MMP-9–/– mice were significantly impaired in their transepithelial migration (Figures 5C and 5D). This was confirmed by CLSM, which showed that a considerable number of MMP-9–/– BMDC remained adherent to the MTE cell monolayer at the end of the 16-h chemotaxis assay (Figure 6A).



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  		Figure 6. MMP-9 deficiency inhibits BMDC migration through the intercellular space. (A) After a 16-h MIP-3ß–induced chemotaxis assay, MTE cell monolayers were immunolabeled for occludin (green) and for MHC class II antigen (red) on BMDC and examined by CLSM. The lower panels are xy sections taken at the level of TJs, and the derived xz sections are shown in each upper panel. The arrows denote the direction of BMDC transepithelial migration. (B) Diagram showing the orientation of the MTE cell monolayers on the filter; the arrows indicate the direction of BMDC transmigration (AP to BL or BL to AP). The dotted line indicates the level of the xy sections. After 16 h some MMP-9–/–, but very few control BMDC, remain on the epithelial cell monolayers. Bar is 80 µm.

 
The Reduced Ability of MMP-9–/– BMDC to Migrate in Response to a Chemotactic Stimulus Is Not Due to Altered Chemokine Receptor Expression
A difference in the level of chemokine receptor expression is a possible mechanism that could account for a reduced response to a chemotactic stimulus. The level of CCR5 (receptor for MIP-1{alpha}) and CCR7 (receptor for MIP-3ß) expression, therefore, was examined by flow cytometry. BMDC from both MMP-9–/– and control mice expressed low, but equivalent amounts of CCR5 (Figure 7). Similarly, although the level of CCR7 expression was considerably higher, equivalent amounts were detected in both MMP-9–/– and control BMDC. Absence of MMP-9 activity in BMDC, therefore, does not impair expression of these chemokine receptors and cannot account for the reduced ability of the MMP-9–/– BMDC to migrate across epithelial cell monolayers in response to a chemotactic stimulus.



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  		Figure 7. Flow cytometric analysis of CCR5 and CCR7 expression by control MMP-9+/+ and MMP-9–/– BMDC. BMDC were isolated from MMP-9–/– mice and their littermate controls and cultured with GM-CSF for 7 d. They were immunolabeled with PE-conjugated anti-CCR5 antibodies. To detect CCR7, BMDC were immunolabeled with CCL19-Ig chimeric protein followed by biotinylated goat anti-human-IgG and PE-streptavidin. Both control MMP-9+/+ (left panels) and MMP-9–/– (right panels) BMDC express similar levels of CCR5 and CCR7. The percent of positively labeled cells is indicated above the horizontal lines.

 
DC Migration into Pulmonary Airspaces Is Impaired in MMP-9–/– Mice
Conditions prevailing in vitro are very different from those in the intact lung. To determine whether our in vitro observations reflect in vivo responses, we used an OVA-induced asthma model to examine the effect of MMP-9 deficiency on DC recruitment into pulmonary airspaces. Analysis of BAL cells in PBS-challenged MMP-9–/– and control mice indicated that there was no significant difference in the number of macrophages, which were the predominant cell type. In OVA-immunized and -challenged mice there was a significant increase in the number of macrophages in BAL fluid as compared with their PBS-treated counterparts (data not shown), but there was no significant difference in their number between control and MMP-9–/– mice. To quantify the DC population in BAL fluid, four-color flow cytometry was performed. DC were identified as low-autofluorescence, CD45R/B220–, CD11c+, CD11b+, I-A/I-E+ cells (Figure 8A). There were fewer DC in the BAL fluid of MMP-9–/– mice as compared with that of MMP-9+/+ control mice after OVA challenge (Figure 8B). These results confirm recently reported data (32) and indicate that MMP-9 is critical for DC transmigration into pulmonary airspaces.



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  		Figure 8. FACS analysis of DC recruitment in vivo. Mice were immunized and challenged with OVA or PBS as described in MATERIALS AND METHODS. Twenty-four hours after the last challenge, BAL cells were harvested and analyzed. (A) Four-color FACS analysis of BAL cells from OVA-treated control MMP-9+/+ (left panel) and MMP-9–/– (right panel) mice. BAL cells were immunolabeled with anti-CD45R/B220-FITC, anti-CD11c–PE, anti-CD11b–APC, and anti–I-A/I-E–biotinylated mAbs, and the latter was incubated with streptavidin-PerCP. To exclude B cells and high autofluorescent macrophages, BAL cells were first gated on CD45R/B220–/CD11c+ cells. This gated population contained a putative CD11b+/I-A/I-E+ DC population, which was quantified. (B) Absolute number of DC in BAL fluids was calculated from the percent of low-autofluorescent, CD45R/B220–, CD11c+, CD11b+, I-A/I-E+ cells multiplied by the total number of viable cells in the BAL fluid. The number of DC in OVA-treated MMP-9–/– mice was lower than that of control OVA-treated mice. Values are mean ± 1 SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During their rapid recruitment in response to inhaled antigens and released chemokine signals, DC must traverse a number of complex tissue compartments. A broad range of factors regulate DC migration, and a number of the mechanisms involved in DC interaction with endothelial cells and connective tissue matrices have been examined (33–35). However, relatively little is known about the mechanisms used by DC to migrate through lung epithelium in response to a chemotactic signal, while at the same time maintaining the integrity of the critically important TJ barrier (1, 36).

In the present study the following two questions were addressed: (i) Do DC express TJ proteins that would enable them to form transient TJs with lung epithelial cells as they migrate through the intercellular space? (ii) Does MMP-9 facilitate the transepithelial migration of DC? To answer these questions we elected to use a well-defined in vitro model in which a mouse tracheal epithelial cell line (26) is plated on either side of collagen-coated tissue culture inserts, to examine either AP to BL or BL to AP migration of DC. This in vitro model enabled us to assess the integrity of the epithelial cell monolayer during transepithelial DC migration by monitoring TER. To address the role of MMP-9 in this process, we isolated BMDC from MMP-9–/– mice and compared their ability to traverse the epithelial cell monolayer with that of BMDC cultured from control MMP-9+/+ mice.

Earlier studies had suggested that DC in the intestine form transient TJs as they sample the bacterial flora in the intestinal lumen (10). These conclusions were based on data showing occludin and claudin-1 mRNA expression and immunolabeled TJ proteins in the cytoplasm of cultured DC following their exposure to bacteria in vitro. We have extended these studies to the lung epithelium and show by Western blotting that DCs express TJ proteins. Furthermore, during their chemokine-induced transepithelial migration, TER is maintained. Although MMP-9–deficient BMDC were significantly impaired in their migration to the intercellular space and through TJs, a small fraction of the starting population successfully traversed the TJ barrier. We report here two novel observations: (i) following migration through the TJ, DC degrade occludin, but not claudin-1; and (ii) DC from MMP-9–/– mice overexpress claudin-1 relative to controls, and the level remains elevated 4 h after migration through the epithelium.

Occludin and the claudins are the integral membrane proteins constituting the tight junction (36). It is interesting, but perhaps not surprising, that these two tetraspan, integral TJ proteins appear to be differentially regulated. Occludin is not necessary for the integrity of TJs because disruption of both occludin alleles by homologous recombination results in embryonic stem cells that differentiate into polarized epithelial cells and form competent TJs (37). The turnover of occludin is regulated, in part, by the ubiquitin/proteosome pathway, which does not appear to be the case for claudin-1 (38). Although the function of occludin in TJs is unclear, there has been speculation that it might transduce signals from the cell surface. Claudins, by contrast, form the characteristic TJ network, as seen in freeze fracture replicas, and are responsible for the ion selectivity of junctions in different tissues (36). The observation that claudin-1 expression is upregulated in DC from MMP-9–/– mice was an unexpected but highly reproducible finding. The relationship between claudins and MMPs is an interesting one and an area that is just beginning to be examined. For example, in the multistep activation of pro–MMP-2, it has been shown that claudins-1, -2, -3, or -5 cannot only substitute for TIMP-2 in the activation of pro–MMP-2, but they can also promote the MMP-14–mediated activation of pro–MMP-2 (39). The direct or indirect contribution of MMP-9 activity to the regulation of claudin-1 expression, however, will require further studies.

To address the second question pertaining to the role of MMP-9 in facilitating the polarized transepithelial migration of DC, we turned to MMP-9–/– mice. Previous in vitro studies had shown that human monocyte-derived DC express MMP-9, and that this enzyme is required for DC migration through cell-free layers of Matrigel (34), but no information was available regarding the role of MMP-9 in DC migration through epithelial TJs. We had originally hypothesized that the direction of transepithelial migration (BL to AP versus AP to BL) might be related to the maturity of the DC, such that immature DC would favor the BL to AP route, whereas more mature DC that re-enter the lung after antigen uptake might prefer the AP to BL pathway. We used MIP-1{alpha} and MIP-3ß as chemoattractants for immature and mature DC, respectively (40). Although control DC migrated in a time-dependent manner across the epithelial cell monolayer, MIP-3ß clearly elicited a much stronger response than MIP-1{alpha}, indicating that BMDC cultured in GM-CSF for 7 d are predominantly a mature DC population (4, 33, 41). This was supported by the observation that CCR7 (receptor for MIP-3ß) was much more strongly expressed than CCR5 (receptor for MIP-1{alpha}). Except for the reduced MIP-1{alpha} elicited BL to AP migration of control BMDC, we did not detect a directional transmigratory preference (BL to AP versus AP to BL) in cultured BMDC from either MMP-9–/– or control mice. This differs from the migratory behavior of neutrophils, which favor the BL to AP migratory route through epithelia (42). However, unlike DC, neutrophils engulf and destroy the offending agent in situ on the mucosal surface and normally do not re-enter the tissue.

These considerations notwithstanding, BMDC from MMP-9–/– mice were significantly impaired in their migration across the epithelial cell monolayer and its TJs. We noted in the course of our confocal microscopy studies that a significant number of MMP-9–/– BMDC remained adherent to the surface of the monolayer even at the end of the 16-h chemotaxis assay. DC express a number of adhesion molecules including CD11a (LFA-1), CD11c, intercellular adhesion molecule (ICAM)-1, -2, and -3, as well as LFA-3 and CD44 (43). Recently JAM-1, a TJ-associated molecule belonging to the immunoglobulin superfamily (44), has been shown to act as a ligand for LFA-1 (45). Both CD44 (23, 46) and ICAM-1 (47) can serve as a docking site for both the pro- and active form of MMP-9, where it is strategically placed to cleave components of the extracellular matrix. It has also been suggested that ICAM-1 on the cell surface may provide a privileged environment protecting active MMP-9 from TIMPs (23, 46). In addition to docking on ICAM-1, MMP-9 can proteolytically cleave ICAM-1 (47). We speculate that localized, controlled cleavage of ICAM-1, and/or other adhesion molecules that serve as focal adhesion sites between DC and the substratum, may be required for DC migration on the surface of epithelial cells. In the absence of MMP-9, therefore, DC migration toward the intercellular space would be impaired. Our observations that cultured MMP-9–/– BMDC adhered to tissue culture plastic, and that significant numbers of DC remained adherent to the MTE-7b cell monolayer after a 16-h incubation, supports such a mechanism.

It has recently been reported that in an OVA-induced model of asthma produced in MMP-9–/– mice, lung DC, but not macrophages, were markedly impaired in their migration into airway and alveolar spaces (32). However, when these investigators instilled CFSE-labeled MMP-9–/– BMDC into the lung or administered FITC-labeled albumin intratracheally, labeled DC were found in local lymph nodes, suggesting that MMP-9 was not required for transjunctional migration. Although comparisons between in vitro and in vivo observations should be made with caution, our in vitro observations are as a whole in agreement with those made in vivo. Thus our studies with BMDC from MMP-9–/– mice indicate that: (i) transepithelial migration is impaired largely due to the reduced ability of MMP-9–/– DC to migrate on the surface of epithelial cells toward the TJs; (ii) the few MMP-9–/– BMDC that successfully traverse the monolayer, however, form competent, transient TJs similar to those of BMDC from control mice; and (iii) our in vitro studies afforded us a means to examine the expression of TJ proteins by BMDC in greater detail and revealed the interesting differential regulation of TJ protein expression following transepithelial migration. Occludin in BMDC is degraded following transmigration, whereas claudin-1 is not; in fact, claudin-1 is significantly upregulated in MMP-9–/– BMDC. Further studies are planned to examine the underlying mechanisms.


   Acknowledgments
 
This study was supported by NIH grants HL25822 and HL36781. The technical assistance of Stacy Francis is gratefully acknowledged. The authors thank Dr. S. Artavanis-Tsakonas for giving us access to the MGH Cancer Center confocal microscopy facility. They thank Dr. Zena Werb for generously providing us with breeding pairs of MMP-9–/– deficient mice, Dr. Merry Lindsey for providing us with a genotyping method, and Dr. Ivan Stamenkovic for stimulating discussions.

Received in original form October 15, 2003

Received in final form December 1, 2003


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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