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Identification of Tissue Transglutaminase as a Novel Molecule Involved In Human CD8⁺ T Cell Transendothelial Migration ¹

Karkada Mohan^*, Devanand Pinto and Thomas B. Issekutz^2,*,

Departments of ^* Pediatrics, Microbiology/Immunology and Pathology, Dalhousie University, and Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
During inflammation, T lymphocytes migrate out of the blood across the vascular endothelium in a multistep process. The receptors mediating T cell adhesion to endothelium are well characterized; however, the molecules involved in T cell transendothelial migration (TEM) subsequent to lymphocyte adhesion to the endothelium are less clear. To identify receptors mediating TEM, mAbs were produced against human blood T cells adhering to IFN--activated HUVEC in mice and tested for inhibition of lymphocyte TEM across cytokine-activated HUVEC. Most of the mAbs were against ₁ and ₂ integrins, but one mAb, 6B9, significantly inhibited T cell TEM across IFN-, TNF-, and IFN- plus TNF--stimulated HUVEC, and did not react with an integrin. 6B9 mAb did not inhibit T cell adhesion to HUVEC, suggesting that 6B9 blocked a novel pathway in T cell TEM. The 6B9 Ag was 80 kDa on SDS-PAGE, and was expressed by both blood leukocytes and HUVEC. Immunoaffinity purification and mass spectrometry identified this Ag as tissue transglutaminase (tTG), a molecule not known to mediate T cell TEM. Treatment of HUVEC with 6B9 was more effective than treatment of T cells. 6B9 blockade selectively inhibited CD4^-, but not CD4⁺, T cell TEM, suggesting a role for tTG in recruitment of CD8⁺ T lymphocytes. Thus, 6B9 is a new blocking mAb to human tTG, which demonstrates that tTG may have a novel role in mediating CD8⁺ T cell migration across cytokine-activated endothelium and infiltration of tissues during inflammation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Extravasation and accumulation of leukocytes in the tissues is a hallmark of chronic inflammatory conditions. Lymphocyte trafficking, both during normal circulation and recruitment to sites of inflammation, involves an intricate multistep process in which lymphocyte surface integrins, endothelial adhesion molecules, and chemokines produced in the local micro environment play an essential role (1, 2, 3). Circulating lymphocytes initially tether and roll on vascular endothelium (endothelial cells (EC)) ³ using the selectins, ₄ integrins, and CD44 (4, 5, 6, 7, 8). These initial interactions are thought to slow the transit of T cells and expose them to activating stimuli, such as surface-bound chemokines on EC (9), and to induce the T cells with activated integrin molecules to undergo arrest and firm adhesion to EC (10, 11). The integrins ₄₁ (very late activation Ag-4 (VLA-4)), ₄₇, and _L2 (LFA-1) have been implicated in activation-dependent stable arrest of T cells (12) through their ability to bind the Ig superfamily adhesion molecules such as ICAM-1, ICAM-2, VCAM-1, and mucosal addressin cell adhesion molecule-1 on the endothelium.

During inflammation, EC are activated by various cytokines such as IFN-, TNF-, and IL-1, which induce expression of adhesion molecules, including E- and P-selectin and VCAM-1, and up-regulate other adhesion molecules such as ICAM-1 (1, 13, 14, 15), thereby contributing to the firm adhesion of T cells onto the endothelium. Although the roles of the selectins, integrins, and their corresponding ligands in the process of T cell adhesion to EC are relatively well characterized, less is known about the molecules participating in the process of transmigration of the endothelium. Transendothelial migration (TEM) by T cells of the vascular wall involves firm adhesion of the cell to the endothelium, followed by diapedesis of the T cells and penetration of the subendothelial basement membrane (16, 17, 18). Previous studies have shown that cytokine activation of EC cannot only increase lymphocyte adhesion to EC, but also T cell TEM across HUVEC monolayers (15, 19, 20, 21). The TEM across HUVEC in response to cytokine activation was shown to be primarily mediated by LFA-1 on the lymphocytes (22, 23, 24). However, TEM in response to several chemokines, including CC chemokine ligand (CCL)5, CCL3, CXC chemokine ligand (CXCL)11, and CXCL12, across cytokine-activated endothelium was only partially inhibited by LFA-1 blockade and was also dependent on VLA-4 (24, 25). Combined blockade of both these integrins virtually abolished T cell TEM in response to CCL5, CCL3, and CXCL11, and markedly reduced migration to CXCL12 across TNF--stimulated HUVEC. These studies suggested that the function of these integrins were essential for migration across the endothelium, but also indicated that other molecular interactions were likely to be important. In addition to integrins, both leukocyte and endothelial CD31 (platelet-endothelial adhesion molecule-1 (PECAM-1)) localized in EC junctions have been shown to be involved in monocyte diapedesis (26).

To identify additional molecules that may mediate T cell TEM, in the present study, mAbs reacting with human blood T cells adhering to IFN--treated HUVEC were produced and tested for their ability to inhibit T cell TEM across cytokine-activated endothelium. In addition to isolating several mAbs against known ₁ and ₂ integrins, a mAb that did not affect T cell EC adhesion, but inhibited T cell TEM across cytokine-stimulated HUVEC, was found. This mAb reacted with tissue transglutaminase (tTG) and was used to demonstrate that tTG played a significant and previously unrecognized role in mediating T cell TEM of a subpopulation of T lymphocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abs and reagents

Recombinant human CXCL11 and CCL5 were obtained from PeproTech (Rocky Hill, NJ), and CXCL12 and TNF- (specific activity = 5 x 10⁷ U/mg) were from R&D Systems (Minneapolis, MN). IFN- (10⁷ U/mg) was from Genentech (South San Francisco, CA). Anti-CD4 (OKT4), anti-CD8 (OKT8), and anti-LFA-1 (IB4) mAbs were obtained from American Type Culture Collection (Manassas, VA). Anti-VLA-4 (HP2/1) was a generous gift from Dr. F. Sanchez-Madrid (Universidad Autonoma de Madrid, Madrid, Spain). Mouse Ab isotyping kit was purchased from Sigma-Aldrich (St. Louis, MO)

Blood lymphocyte isolation

Lymphocytes were isolated from human peripheral blood of healthy donors by gradient centrifugation and passaged over a nylon wool column as previously described (15). In brief, acid citrate dextrose-heparin anticoagulated blood was gently mixed with an equal volume of warm PBS, layered onto Ficoll-Paque (Pharmacia, Uppsala, Sweden), and centrifuged at 900 x g for 20 min. The PBMC on top of the Ficoll-Paque were collected and washed three times with Ca²⁺ Mg²⁺-free Tyrode’s solution. The PBMC were resuspended in RPMI 1640 medium with 10% human platelet-poor plasma and were applied onto a nylon wool column. After 60 min of incubation, the unbound T lymphocytes were eluted, washed, resuspended in fresh RPMI medium plus 10% plasma at 2 x 10⁶ cells/ml, and cultured overnight in tissue culture flasks. Previous experiments have shown that this overnight culture of T cells does not induce the expression of activation markers and does not appear to alter the mechanism of T cell migration, although it causes a small increase in total migration, which may be caused by resting of the cells after the isolation procedure. The nonadherent cells contained >96% T cells, <3% B cells, and <0.1% monocytes by immunofluorescence staining, and were >98% viable by trypan blue dye exclusion.

In some studies, T cells enriched in CD4⁺ or CD8⁺ T cells were isolated using MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, T cells were incubated with mAb to CD4 (OKT4) or CD8 (OKT8) at 50 µg/10⁸ cells/ml in RPMI medium plus 10% FCS at 4°C for 30 min. The cells were then washed twice and resuspended in HEPES-buffered HBSS containing 10% FCS at 10⁷ cells/90 µl medium. A total of 10 µl of goat anti-mouse Ig-conjugated-magnetic beads was added, and the suspension was incubated at 6–12°C for 15 min, with mixing every 5 min. After an additional wash, the cells were placed on the MACS, and the cells not adhering to the column were collected as CD4^- cells or CD8^- cells, respectively. The column was washed repeatedly, and the adherent cells eluted as the CD4⁺ and CD8⁺ cells. The purity was >99% for CD4⁺ and CD8⁺ T cells by immunofluorescence staining.

Isolation and culture of EC

HUVEC were isolated by collagenase digestion as described (27). Briefly, human umbilical veins were flushed with Ringer’s Lactate, then incubated with 0.5 mg/ml collagenase type II (Sigma-Aldrich) at 37°C for 30 min. Detached EC were collected, washed, and cultured in gelatin-coated flasks (Nunc, Naperville, IL) in RPMI containing 20% FBS (HyClone Laboratories, Logan, UT), 25 µg/ml EC growth supplement (BD Biosciences, Bedford, MA), 45 µg/ml heparin, 2 mM L-glutamine, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. The HUVEC in these studies was used only up to passage three.

Measurement of lymphocyte TEM

Confluent HUVEC grown in flasks were gently trypsinized and seeded onto polycarbonate microporous membranes of 6.5 mm diameter and 5 µm pore size in Transwell Chambers (Costar, Cambridge, MA) that had been coated with 0.01% gelatin at 37°C overnight, followed by 3 µg of human fibronectin (Fn) (Life Technologies, Grand Island, NY) at 37°C for 3 h. HUVEC (1.2 x 10⁴) were added to each Transwell in 100 µl of HUVEC medium, and 0.6 ml of the same medium was added to the lower chamber beneath the Transwell. After 6 days of culture, the integrity of confluent HUVEC monolayers was assessed by microscopic observation and by measuring the permeability of the monolayer using ¹²⁵I-labeled albumin diffusion.

T lymphocyte migration was measured as previously described (15). T cells were labeled by incubating 5 x 10⁷ cells per milliliter in RPMI plus 10% FBS with 50 µCi/ml Na₂⁵¹CrO₄ (Amersham, Oakville, Canada) at 37°C for 45 min. Cells were washed three times with RPMI and resuspended in RPMI plus 5 mg/ml human serum albumin. The HUVEC monolayers in the Transwells were either left untreated or were pretreated by incubation with TNF- (200 U/ml), IFN- (200 U/ml), or IFN- plus TNF- added to the lower chamber beneath the monlayer for 18 h. The endothelial monolayers in the Transwell inserts were gently rinsed with RPMI, and 100 µl of labeled T cells (2 x 10⁵ cells) were added on top of the HUVEC monolayers. The inserts were transferred to a new 24-well plate (lower chambers) containing 0.6 ml of fresh RPMI plus human serum albumin, with or without added chemokine, and incubated at 37°C in 5% CO₂. After 4 h, T cells that had migrated through the HUVEC monolayers into the lower chamber of each well were recovered, and the radioactivity in these samples was determined by gamma counting. The percentage of migrated cells was calculated by dividing the radioactivity of cells in the lower chamber by the total input radioactivity on the T cells added to the upper chamber. Spontaneous release of ⁵¹Cr from the labeled cells during the 4-h migration assay was <2%.

In Ab blocking experiments, T cells were pretreated with 20 µg/ml of the test or control mAb for 20 min, and then added to the Transwells on top of the EC monolayers without the mAbs being removed. In some assays, T cells and the HUVEC monolayer on the Transwell cups were separately pretreated with mAbs for 30 min and washed using RPMI medium to remove unbound mAbs. Lymphocyte migration was measured after 90 min to minimize Ag re-expression on the surface of the cells.

Measurement of lymphocyte chemotaxis

T cell migration in response to chemokines was measured using 5-µm pore size Transwell chambers as described for TEM, except that no HUVEC was added to the membranes coated with gelatin and Fn. Chemokines were added to the lower chamber of each well, and the plates were incubated for 60 min at 37°C in 5% CO₂. The migrated T cells in the lower chambers were collected, and the extent of T cell migration was measured by gamma counting and calculated as above.

Production of mAbs

Human blood T cells were isolated and allowed to adhere to confluent monolayers of HUVEC treated with IFN- in 100-mm tissue culture dishes. After 1 h the nonadherent lymphocytes were removed by washing, and the adherent lymphocytes and EC were detached with cold EDTA and gentle agitation. BALB/c mice were repeatedly immunized with this cell suspension. Three days after the last immunization, the spleens of the mice were removed. The splenocytes were fused with a nonsecreting mouse myeloma, P3U1, using polyethylene glycol 4000 (Life Technologies, Burlington, Canada) and plated in 96-well plates in DMEM plus 10% FBS, antibiotics, hypoxanthine, aminopterin, and thymidine. After 2 wk, the supernatants from the hybridomas were screened for their ability to inhibit the TEM of blood T lymphocytes across cytokine-activated HUVEC in Transwell chambers. Wells containing hybridomas that repeatedly tested positive were cloned by limiting dilution. Ab isotyping was done using a mouse Ab isotyping kit (Sigma-Aldrich) according to the manufacturer’s instructions.

Immunofluorescence staining

Briefly, cells were washed, resuspended in PBS with 0.5% BSA plus 0.1% NaN₃, and incubated with 10 µg/ml mouse mAb at 4°C for 30 min. Cells were washed twice and incubated with FITC-conjugated sheep anti-mouse IgG (Sigma-Aldrich). Finally, cells were washed, fixed in 1% paraformaldehyde in PBS, and analyzed by flow cytometry using a FACSCalibur (BD Biosciences).

Measurement of lymphocyte adhesion

HUVEC were grown to confluence in gelatin-coated 96-well tissue culture plates. Cells in the monolayer were left untreated or were stimulated by adding TNF- (200 U/ml), IFN- (200 U/ml), or IFN- plus TNF- (200 U/ml each) for 18 h. ⁵¹Cr-labeled T lymphocytes (2 x 10⁵ cells in 100 µl RPMI plus 10% FCS), treated with 20 µg/ml of control or test mAb for 20 min at room temperature, were added to triplicate wells. The cells were allowed to adhere at 37°C for 60 min. Nonadherent cells were removed by four washes with warm RPMI. The bound T cells were lysed with 0.1 N NaOH and collected into tubes, and the radioactivity was measured by gamma counting. The percentage of cell adhesion was calculated by dividing the radioactivity of bound cells by the radioactivity of total input cells.

Immunoprecipitation

Human blood T cells, the HUT-78 T cell line, and HUVEC were surface biotinylated using long chain biotin (Pierce, Rockford, IL). Briefly, cells were washed three times with cold PBS and resuspended at 10⁷ cells/ml in Ca²⁺/Mg²⁺-supplemented PBS, and biotin was added to a final concentration of 0.5 mg/ml. Cells were kept on ice for 2 h with gentle mixing followed by two washes in PBS and one wash in 1 mM diisopropyl fluorophosphate in PBS. Cells (2 x 10⁷ cells/ml) were lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 mM DTT, 5 µg/ml pepstatin and leupeptin, and 1 µg/ml aprotinin. Lysis was conducted for 30 min on ice with repeated mixing, followed by centrifugation at 21,000 x g for 30 min at 4°C to remove insoluble debris. One-fifth volume of NaDOC was added to the supernatant, and after 10 min on ice, the lysate was again centrifuged as above. The cell lysates were precleared with protein G-Sepharose 4B beads and goat anti-mouse IgG immobilized on protein G beads overnight at 4°C. One-tenth volume of hybridoma supernatants was added to the lysates, and after a 2-h incubation at 4°C, Ag-Ab complexes were captured by adding goat anti-mouse IgG-coupled protein G-Sepharose 4B beads to the mixture and rotating for 1.5 h at 4°C. Protein G beads with captured Ag were washed four times with PBS plus 0.5% Triton X-100 before the immunoprecipitates were eluted using SDS-sample buffer. Precipitated Ags were separated by SDS-PAGE on a 10% gel and transferred onto PVDF membranes, followed by the membrane being quenched with 5% skimmed milk in PBS containing 0.2% Tween 20 for 1 h. The membrane was washed four times with PBS with 0.2% Tween 20 before being incubated with HRP-streptavidin (Pierce) for 1 h. Following additional washes of the membrane, enzymatic development was performed by using the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ).

Affinity purification and mass spectrometry (MS)

To identify the Ag recognized by the 6B9 mAb using MS, affinity purified 6B9 Ag was obtained using Ab-conjugated Sepharose beads according to published methods (28). Briefly, a lysate of 1.2 x 10⁹ HUT-78 cells was prepared (10⁸ cells/ml lysis buffer) essentially as above, but without surface biotinylation. The cell lysate was precleared using 0.1 volume of glycine-quenched Sepharose twice for 2 h and once overnight at 4°C. A negative control mAb, RMP-1, and the 6B9 mAb were covalently conjugated to cyanogen bromide-activated Sepharose 4B beads (Pharmacia) according to the manufacturer’s instructions. The cell lysate, diluted in 10 mM Tris (pH 7.5), was passed over a column of the mAb-coupled beads, and the columns were washed with 20-column volumes of wash buffer (10 mM Tris, 140 mM NaCl, 0.5% Triton X-100, and 0.025% NaN₃, pH 8.0) and a buffer of 50 mM Tris Cl pH 8.0, and pH 9.0. Ab-bound Ag was eluted using a five-column volume of 50 mM triethanolamine with 150 mM NaCl and 0.5% Triton X-100 (pH 11.5), and fractions of one-column volume in size were collected into tubes containing 0.2 vol of 1 M Tris (pH 6.7). The affinity purified Ag was precipitated with 5 vol of cold acetone for 18 h at -20°C; proteins were pelleted and redissolved in SDS sample buffer. The purified Ag was then analyzed on a 10% SDS-PAGE gel and silver stained using a standard protocol. The 6B9 affinity purified Ag was excised from the gel and subjected to in-gel digestion using trypsin as previously described (29). The tryptic peptides were injected onto a 100 µm x 5 cm HPLC column packed with 5-mm Spherisorb ODS-2 C₁₈ particles. The column contained an integrated nanospray tip for interfacing to the MS (30). MS spectra and tandem MS spectra were collected using a prototype Qstar MS (Qq-ToF geometry) from MDS Sciex (Thornhill, Ontario, Canada). Tandem MS spectra were searched against the National Center for Biotechnology Information database using the software program MASCOT (Matrix Science, London, U.K.).

Statistical analysis

Data are expressed as either the mean ± SEM of multiple assays or as the mean ± SD of triplicate determinations from a representative experiment. ANOVA and Student’s unpaired t test were used to compare the differences between means, as appropriate.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of mAbs inhibiting T cell TEM

During inflammation, T cells migrate across cytokine-activated vascular endothelium. The present study was undertaken to identify novel molecules participating in the process of T cell TEM across cytokine-activated endothelium. Previously we showed that IFN- was a potent stimulator of human blood T cell TEM. Therefore, mice were immunized with T cells adhering to IFN--stimulated endothelium to generate mAb that could recognize molecules taking part in T cell-endothelial interaction during T cell TEM (15). Fig. 1 shows that treatment of HUVEC with IFN- plus TNF- increased T cell TEM by >5-fold, from 4% across unstimulated HUVEC to 23% across the activated HUVEC. This in vitro model of T cell TEM was used to identify hybridomas that produced mAbs capable of inhibiting T cell TEM. At least 10 mAbs that significantly inhibited T cell TEM by 40–60% were identified. Further analysis using immunoprecipitation showed that most, but not all, of these mAbs were against previously described integrin molecules, such as LFA-1 and ₁ integrins (data not shown). One of the mAbs, 6B9, consistently inhibited the increased cytokine-stimulated T cell TEM by 40%. As shown in Fig. 1, the 6B9 mAb decreased T cell migration from 23% to 15% of input T cells. This mAb was further characterized to identify the Ag that it recognized. The 6B9 mAb was found to be of the IgG₁ subclass.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Effect of mAb 6B9 on TEM of T cells across cytokine-activated HUVEC. Confluent monolayers of HUVEC grown on Transwell membranes were unstimulated or stimulated with 200 U/ml IFN- plus TNF- for 18 h. 51Cr-labeled T cells were either untreated or treated with 20 µg/ml control mAb B9 or mAb 6B9 for 30 min, added to the upper chamber of the Transwell, and allowed to migrate across the HUVEC for 4 h into the lower chamber. The percentage of migration was calculated by dividing the radioactivity of migrated cells by the total radioactivity of the cells added to the upper chamber. Each bar shows the mean ± SEM of nine independent experiments using HUVEC and T cells from different donors, each with triplicate determinations. x, p < 0.01.

To examine the cell surface expression of the Ag reacting with 6B9 mAb, human blood leukocytes, EC, and fibroblasts were stained by immunofluorescence with 6B9. As shown in Fig. 2, the 6B9 mAb stained all of the cell types tested, including virtually all blood lymphocytes, monocytes, and neutrophils, as well as the HUVEC and fibroblasts, indicating that the 6B9-recognized Ag was expressed on a wide array of cell types. The 6B9 mAb did not stain mouse or rat leukocytes (data not shown). In some experiments, expression of the 6B9 Ag on HUVEC activated with IFN-, TNF-, or both of these cytokines was examined by immunofluorescence staining and compared with unstimulated HUVEC. 6B9 stained virtually all of the HUVEC, whether unstimulated or cytokine activated, but the mean fluorescence intensity was twice as high on IFN- plus TNF--activated HUVEC as on unstimulated EC (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Immunofluorescence staining of blood leukocytes, HUVEC, and fibroblasts with mAb 6B9. Blood leukocytes from normal donors were isolated and without culture used for immunofluorescence staining. These cells and suspensions of HUVEC and fibroblasts were stained using control mAb B9 (dashed line) or mAb 6B9 (gray filled), followed by FITC-conjugated sheep anti-mouse Ig, and analyzed by flow-cytometer.

To determine the molecular mass of the Ag to which the 6B9 mAb binds, blood lymphocytes, the lymphoblast cell line, HUT-78, and HUVEC were surface biotinylated, and cell lysates were prepared. These lysates were incubated with the 6B9 mAb, an isotype-matched control mAb (B9), or a mAb to LFA-1 (3A12). The Ags immunoprecipitated by these mAbs were analyzed on SDS-PAGE. Fig. 3 shows a representative precipitation. The anti-LFA-1 mAb immunoprecipitated the expected -(180 kDa) and -(95 kDa) chains of this integrin from the blood lymphocytes; whereas the 6B9 mAb precipitated a protein with an approximate molecular mass of 80 kDa. Immunoprecipitation of both the HUT-78 and HUVEC with 6B9 yielded very similar results.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Immunoprecipitation of blood T cells with the 6B9 mAb. T cells were surface biotinylated using long chain biotin, lysed with Triton-X, and immunoprecipitated with either control mAb B9, anti-LFA-1 mAb (3A12), or the 6B9 mAb. Ag-Ab complexes were captured with anti-mouse Ig-coated Sepharose beads. The Ags were eluted in SDS sample buffer, separated by SDS-PAGE, transferred to nitrocellulose, and detected using streptavidin-HRP and the ECL system. The column on the right shows molecular mass markers in kilodaltons.

To identify the protein to which 6B9 bound, the 6B9 Ag was affinity purified by passing a cell lysate from HUT-78 cells through a column of 6B9 mAb-coupled Sepharose beads and eluting the Ag at high pH. Fig. 4 shows that analysis of the eluted material by silver staining on SDS-PAGE revealed a major band at 80 kDa, similar to the immunoprecipitations. This 80-kDa band was excised from the gel, trypsin digested to examine peptide construction, and used in MS analysis. The tandem MS spectra were searched against the National Center for Biotechnology Information database using MASCOT software (Matrix Science). A peptide with a molecular mass of 1610.80 Da was matched to the 15-residue fragment TVEIPDPVEAGEEVK. The peptide mass correlated well with the measured mass (1610.86 Da), and the MASCOT score was 53 (scores of >42 indicate identity). This peptide with 15 aa residues was used in GenBank protein blast searches (31). A 100% match of this peptide was obtained with human tTG, also called type 2 transglutaminase, indicating that the 6B9 Ag was tTG, which is an 80-kDa protein.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Immunoaffinity purification of the Ag bound by the 6B9 mAb. Cell lysate from 1.2 x 109 HUT-78 cells were passed through columns containing either control mAb (RMP1) or mAb 6B9-conjugated Sepharose 4B and eluted using 50 mM triethanolamine (pH 11.5), as described in Materials and Methods. Purified Ags were run on SDS-PAGE and silver stained. The 80-kDa 6B9 Ag was cut out and trypsin digested. The resulting peptides were analyzed by MS.

Because these results are the first to show that an Ab to tTG could inhibit T cell TEM, further investigations were performed to examine the role of tTG in this process. The adhesion of leukocytes to EC constitutes one of the critical steps leading to TEM by leukocytes. To determine whether tTG was required for T cell adhesion to HUVEC, the ability of T cells to adhere to unstimulated and cytokine-activated HUVEC was examined in the presence and absence of the 6B9 mAb. As shown in Fig. 5, 5.7% of T cells adhered to resting EC, and IFN-, TNF-, and IFN- plus TNF- treatment of the HUVEC significantly increased T cell adhesion to 16.6, 34.7, and 40.8%, respectively. The 6B9 mAb did not have any effect on T cell adhesion to the cytokine-activated HUVEC, even though this same concentration of 6B9 significantly inhibited T cell TEM. This suggested that tTG was not required for T cell adhesion to HUVEC, and that the effect of the 6B9 mAb on TEM was not through an action on T cell adhesion to the EC.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Effect of the 6B9 mAb on the adhesion of T cells to cytokine-activated HUVEC. Confluent monolayers of HUVEC in 96-well plates were either left untreated or stimulated with 200 U/ml IFN- or TNF- or both cytokines for 18 h. 51Cr-labeled T cells were either left untreated or treated with 20 µg/ml control mAb B9 or 6B9 mAb for 30 min, and added to the EC monolayers. The T cells were allowed to adhere for 1 h at 37°C. Nonadherent T cells were removed by washing, and the percentage of cell adhesion was determined by gamma counting of the adherent T cells. Each bar represents the mean ± SEM of four to six independent assays, each done in triplicate using different donor cells.

Chemokines can stimulate an increase in integrin affinity on T cells and can stimulate T cell migration along a chemokine concentration gradient. Chemokines are up-regulated at sites of inflammation by proinflammatory cytokines, such as IFN- and TNF-, and direct recruitment of T cells across the endothelium into the tissue. Because cytokine treatment of the HUVEC will increase chemokine production by the endothelium, the effect of mAb 6B9 on the chemotaxis of blood T cells to chemokines across gelatin plus Fn-coated microporous membranes was examined (Fig. 6A). Spontaneous migration across these membranes was 4% of input T cells; and the chemokines CXCL12, CXCL11, and CCL5 increased T cell chemotaxis to 50, 20, and 15% of the T cells, respectively (Fig. 6A). The 6B9 anti-tTG mAb had no effect on the chemotaxis induced by the three chemokines.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Effect of the 6B9 mAb on blood T cell chemotaxis and TEM. 51Cr-labeled T cells were either left untreated or treated with control mAb B9 or mAb 6B9, and their migration in response to CXCL12 (100 ng/ml), CXCL11 (200 ng/ml) and CCL5 (200 ng/ml) was determined across gelatin/Fn-coated Transwell membranes (bare membrane chemotaxis) after 1 h (A), and across unstimulated HUVEC monolayers in response to chemokines after 4 h (B). Spontaneous T cell migration in the absence of chemokines was 3–4%. Values shown are the percentage of total input radiolabeled T cells, and each bar represents the mean ± SEM of four to six independent experiments, each with triplicate determinations using HUVEC and T cells from different donors.

Role of tTG in T cell TEM across cytokine-activated endothelium

To determine the effect of the tTG blockade with mAb 6B9 on T cell TEM across cytokine-activated EC, HUVEC were treated with IFN-, TNF-, or both cytokines for 18 h, and T cell TEM was determined in the presence or absence of the anti-tTG mAb. As shown in Fig. 7, T cell migration across HUVEC treated with IFN-, TNF-, or both cytokines was increased 4- to 6-fold, from 3.5% spontaneous migration to 16–23%. An isotype control mAb had no effect on T cell TEM, but the 6B9 mAb significantly inhibited T cell TEM across all three cytokine-treated EC to 12.9, 13.4, and 15.9%, resulting in a 28, 42, and 34% inhibition of migration to HUVEC treated with IFN-, TNF-, and both cytokines, respectively. Interestingly, although T cell TEM to chemokines across unstimulated HUVEC was not affected by blockade of tTG (Fig. 6B), migration of T cells in response to CXCL12 across cytokine-activated HUVEC was significantly inhibited by anti-tTG mAb (p < 0.02, data not shown). These observations suggested that cytokine activation of endothelium with IFN- or TNF- was required for tTG to play a role in T cell TEM.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Effect of the 6B9 mAb on T cell TEM across cytokine-activated HUVEC. EC grown in Transwell chambers were stimulated with the indicated cytokines for 18 h. 51Cr-labeled blood T cells were left untreated or treated with control mAb B9 or mAb 6B9, and their migration across the HUVEC was determined after 4 h, as outlined in Fig. 1. Each bar represents the mean ± SEM of 5–10 independent experiments, each done in triplicate, using T cells and HUVEC from different donors. Spontaneous migration across unstimulated HUVEC was 3.5%. +, p < 0.05.

Immunofluorescence staining with mAb 6B9 showed that tTG was expressed on both T cells and HUVEC (Fig. 2). To determine whether the inhibition of TEM by mAb 6B9 was through blockade of tTG on the T cell or on the EC, or both, T cells and HUVEC were each treated separately with 6B9. After excess mAb was washed away, T cell TEM across cytokine-activated HUVEC was determined after migration for 90 min. As shown in Fig. 8, treatment of both T cell and HUVEC with 6B9 significantly (p < 0.05) inhibited by 35% T cell TEM, similar to the results in Fig. 7. However, when only the T cells, and not the EC, were treated with 6B9, no significant inhibition of TEM was observed, although the mean TEM was decreased by 20% compared with control T cells. In contrast, treatment of only the HUVEC with the 6B9 mAb significantly (p < 0.01) inhibited T cell TEM; decreasing TEM by >40% compared with control Ab-treated HUVEC. This suggested that tTG on the endothelium likely plays the more important role in mediating T cell TEM.

View larger version (30K): [in this window] [in a new window]   FIGURE 8. Effect of treating either T cells or HUVEC separately with mAb 6B9 on T cell TEM. HUVEC monolayers were stimulated with IFN- plus TNF- for 18 h, and the TEM migration of 51Cr-labeled T cells was determined in Fig. 1. T cells and HUVEC were each treated separately with 6B9 or control mAb for 30 min and washed, and the migration across cytokine-activated HUVEC was determined after 90 min. Values are expressed as the percentage migration relative to the no mAb control. Each bar shows the mean ± SD of triplicate determinations, from one of three similar experiments. +, p < 0.05; and x, p < 0.01.

Blood T lymphocytes, which migrate across cytokine-activated HUVEC, include both CD4 and CD8 T cells and are primarily of the memory (CD45RO⁺) phenotype (15, 25). To determine whether tTG has a differential role in mediating TEM of CD4 or CD8 T cells, blood T lymphocytes were separated into CD4⁺ (CD8^-) and CD4^- (CD8⁺) fractions, and their chemotaxis to CXCL12 and TEM across IFN- plus TNF--activated HUVEC was determined and compared with unfractionated T cells. CXCL12 induced the chemotaxis of 50–60% of unfractionated and CD8⁺ T cells and 70% of CD4⁺ T cells. Treatment with mAb 6B9 did not affect this migration (data not shown), similar to the results in Fig. 6. As shown in Fig. 9, 28% of input unfractionated T cells, 20% of CD4 T cells, and 24% of CD8 T cells migrated across cytokine-activated HUVEC. The anti-tTG mAb 6B9 significantly (p < 0.05) inhibited TEM of unfractionated T cells and had no significant effect on CD4 T cell migration, but it strongly (p < 0.002) inhibited the TEM of CD8 T cell by almost 50%. This suggested that tTG has an important role in mediating transmigration of CD8, but not CD4, T cells across cytokine-activated endothelium.

View larger version (27K): [in this window] [in a new window]   FIGURE 9. Effect of mAb 6B9 on CD4+ and CD4- T cell TEM. Blood T cells were negatively selected for CD8 T cells and separated into CD4+ (CD8-) and CD4- (CD8+) fractions using MACS separation as described in Materials and Methods, and labeled with 51Cr. The T cells were either left untreated or were treated with control mAb B9 or mAb 6B9, and their migration across IFN- plus TNF--activated HUVEC was determined after 4 h. Each bar shows the mean ± SD of triplicate determinations from one of three similar experiments. +, p < 0.05; and x, p < 0.002.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

tTG is one member of a family of transglutaminases comprised of Ca²⁺-dependent enzymes that catalyze the post-translational cross-linking of proteins by forming amide bonds between glutamines and -amino groups of lysine residues (32). At least five distinct forms of transglutaminases have been identified, four of which are mainly intracellular (TGase1–TGase4) and one extracellular form, coagulation factor XIIIa. tTG has been implicated in the following: adhesion of cells to extracellular matrix (ECM) (33, 34), signaling as a GTPase (35, 36), and apoptosis (37, 38). tTG has also been implicated in the pathogenesis of celiac disease (39), neurodegenerative diseases (40, 41), and in atherosclerosis (42, 43). The role of tTG in cell adhesion appears to relate to its ability to function as an integrin-binding coreceptor for Fn (34, 44, 45) and a stabilizer of ECM proteins, by inducing Fn monomer assembly and cross-linking ECM proteins (33, 46, 47, 48, 49, 50, 51).

The present study describes a new mAb that reacts with human tTG and blocks T lymphocyte TEM across cytokine-activated endothelium. This mAb demonstrates high-level expression of tTG on a wide variety of cells, including lymphocytes, monocytes, neutrophils, EC, and fibroblasts; it immunoprecipitates tTG from T cells and human EC. This is the first study that shows that tTG can mediate TEM of T cells, and attempts to dissect the major features of this migration. Previous reports have demonstrated that tTG can associate with ₁ and ₃ integrins to enhance cell adhesion (34, 44, 52). tTG can bind with high affinity to the 42-kDa fragment of Fn, thereby providing additional binding sites for cell adhesion. In addition, the integrin ₄₁ has been shown to bind to immobilized tTg, and it has been suggested that lymphocytes and monocytes that express high levels of ₄₁ may use this for adhesion (44). Akimov and Belkin (45) have also reported that migration of monocytes on Fn is partly mediated by tTG.

Our results suggest that tTG is involved in mediating T cell TEM, but that the effect of mAb 6B9 to decrease TEM is independent of lymphocyte EC adhesion. Several observations support this conclusion. The 6B9 mAb did not inhibit T lymphocyte adhesion to unstimulated or cytokine-activated HUVEC, as shown in Fig. 5. Treatment with mAb 6B9 also did not affect chemotaxis of T cells across Fn or unstimulated HUVEC (Fig. 6), both of which require T cell adhesion. In addition, mAb 6B9 added to T cells that had been allowed to preadhere to cytokine-activated EC were also inhibited from migrating across the HUVEC monolayers by the anti-tTG mAb (data not shown). In contrast, adhesion blocking Abs to LFA-1 or ₁ had much less effect on TEM of T cells that had been allowed to preadhere compared with T cells that had been treated with mAbs before adhesion to the EC monolayers. Thus, tTG does not only promote adhesion to ECM as previously reported (34, 44, 45), it can also mediate TEM through an adhesion-independent mechanism. This ability of tTG to mediate TEM may relate to the increased accumulation of tTG at EC junctions that are sites through which T cells are known to migrate (52).

Another interesting and unexpected finding is the specific effect of a 6B9 mAb blockade on the migration of CD8 T cells across cytokine-activated EC when the mAb was bound to the EC tTG. In the absence of cytokine-activated EC, 6B9 binding to T cells did not inhibit T cell chemotaxis across Fn-coated membranes, and the mAb did not inhibit migration across unstimulated EC (Fig. 6). However, TEM induced by treatment of HUVEC with IFN-, TNF-, or both cytokines was significantly inhibited by mAb 6B9, and this inhibition of migration was primarily of CD8 T cells (Figs. 8 and 9). The basis for this specific effect of the anti-tTG 6B9 on CD8 T cell TEM across IFN- and TNF--activated EC is not clear. tTG expression was found to be increased on HUVEC-treated IFN- plus TNF- in our studies, and TNF- has been reported to enhance tTG activity accompanied by increased multimerization of Fn in the ECM of TNF--treated calf pulmonary artery EC (46). However, the anti-tTG mAb also inhibited TEM across IFN--treated EC, which did not express increased tTG. These findings suggest that the level of tTG on cytokine-activated EC is not related to the ability of tTG to mediate CD8 T cell TEM. Previous reports have also examined the relationship of tTG enzymatic activity to its ability to enhance cell adhesion to ECM. The effect of tTG as an adhesion coreceptor is clearly independent of its enzymatic activity and appears to relate to its ability to associate with integrins (34, 44, 45, 53, 54). Although the enzymatic effects of tTG on the ECM of cytokine-activated EC are likely to occur over many hours, and the effects of cytokine activation of EC are not necessarily dependent upon enzymatic activity, it is unlikely that inhibition of TEM observed in the presence of mAb 6B9 within 90 min (Fig. 8) is dependent upon tTG enzymatic activity. Further studies to specifically block tTG enzymatic activity will be needed to confirm that activity of the enzyme is not required for TEM by CD8 T cells across cytokine-activated EC.

The explanation for the specific inhibition of CD8, but not CD4, T cell TEM by anti-tTG mAb across cytokine-activated EC is unclear. It has been shown that nonactivated resting CD8 T cells express inflammation-associated chemokine receptors and respond to the ligands of these receptors without preactivation, much better than CD4 T cells (55). This may allow these CTLs to invade inflamed tissues earlier than CD4 T cells. These resting T cells may also use additional mechanisms, such as tTG on cytokine-activated endothelium, to transmigrate these inflamed venules. Further studies to characterize the subset of CD8 T cells involved in tTG-dependent TEM may be useful.

In conclusion, our results have described a new mAb to tTG, and suggest that tTG is involved in CD8 T cell TEM through an interaction between the T cell and tTG on cytokine-activated endothelium. These findings may be important in the infiltration of CD8 T cells into inflamed tissues. Further studies of the role of tTG in T cell migration in vivo are essential to clarify the importance of this molecule in lymphocyte migration in inflammation.

	Footnotes

 
¹ This work was supported by the Arthritis Society (Grant TAS89/0002) and the Canadian Institutes of Health Research (Grant MOP-42379). K.M. was supported by a Cancer Research and Education Nova Scotia trainee award with funding from Cancer Care Nova Scotia.

² Address correspondence and reprint requests to Dr. Thomas B. Issekutz, Division of Immunology, Rheumatology and Infectious Diseases, Department of Pediatrics, IWK Health Center, 5850 University Avenue, Halifax, Nova Scotia, Canada, B3J 3G9. E-mail address: thomas.issekutz{at}dal.ca

³ Abbreviations used in this paper: EC, endothelial cell; tTG, tissue transglutaminase; Fn, fibronectin; ECM, extracellular matrix; TEM, transendothelial migration; VLA-4, very late activation Ag-4; PECAM-1, platelet-endothelial adhesion molecule-1; MS, mass spectrometer/spectrometry; CCL, CC chemokine ligand; CXCL, CXC chemokine ligand.

Received for publication March 28, 2003. Accepted for publication July 15, 2003.

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