Published online before print July 26, 2007
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* Cancer and Molecular Immunology Group, Division of Molecular Bioscience, The John Curtin School of Medical Research, Australian National University, Acton, Australia;
Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA; and
Neurosciences Research Unit, The Canberra Hospital, Woden, Australia
1 Correspondence: Cancer and Molecular Immunology Group, Division of Molecular Bioscience, The John Curtin School of Medical Research, The Australian National University, Acton, ACT 2601 Australia. E-mail: mark.hulett{at}anu.edu.au
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Key Words: migration • inflammation • autoimmunity • extracellular matrix • multiple sclerosis
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HPSE cleaves heparan sulfate (HS) chains in the BM and in the ECM, aiding the migration of cells through these structures during injury, inflammation, and tumor metastasis [4 5 6 7 ]. Models of delayed-type hypersensitivity (DTH) in mice have provided indirect evidence of the promigratory role of HPSE. Antigen-specific T lymphocytes pretreated with heparin display an impaired ability to migrate to the site of antigen and cause DTH, an effect that is not observed if the cells are injected directly into the site of the antigen [17 ]. In addition, cleavage of HS chains in the ECM by HPSE results in the release of depots of chemokines and cytokines, which upon activation play an important role in guiding the direction of migrating cells in tissues. Recent studies suggest an additional role for HPSE in T cell migration, namely, as a proadhesion molecule [18 ]. Under shear flow conditions, HPSE was shown to capture T cells and mediate their rolling and arrest.
Consistent with this promigratory function for HPSE, there is growing evidence that HPSE plays an important role in T cell-mediated inflammatory diseases, as demonstrated in animal models, such as experimental autoimmune encephalomyelitis (EAE) [19 20 21 22 ], adjuvant arthritis (AA) [21 ], and survival of skin allografts [21 ]. EAE is a T cell-mediated inflammatory disease of the central nervous system and represents an excellent model of the inflammatory stage of the autoimmune demyelinating disease multiple sclerosis [23 ]. EAE is caused by the infiltration of antigen-specific T lymphocytes into the central nervous system (CNS). A key factor in the development of the disease is the ability of the lymphocytes to penetrate the walls of blood vessels and the subendothelial basement membrane and accumulate in the CNS. A role for HPSE in EAE was first suggested by Naparstek and colleagues, supported by the observation that HPSE activity in antigen-specific T lymphocytes in vitro correlated with the ability of the cells to invade the CNS and cause disease [8 ]. Since then, studies have also correlated the ability of sulfated polysaccharides to prevent disease in vivo with their ability to inhibit HPSE activity in vitro [19 20 21 22 ]. A recent study has suggested that the endothelium is also an important source of heparanase in delayed-type hypersensitivity (DTH)-associated inflammation [24 ].
The tight regulation of HPSE expression and activity in lymphocytes is clearly of critical importance to control erroneous tissue damage and cell migration that may contribute to the development of T cell-mediated diseases. Previous studies in rat and human T cells have shown that HPSE activity is induced upon cell activation [8 ,25 ,26 ], but little is known of the molecular mechanisms that regulate HPSE expression both physiologically and during T cell-mediated disease. Recently, the cloning of HPSE has provided the tools to investigate the molecular basis of inducible HPSE expression [27 28 29 30 31 ]. The present study explores the regulation of HPSE mRNA and protein expression both in vitro and in vivo in the T cell-mediated disease model, EAE. We show that HPSE mRNA and protein expression in CD4+ antigen-specific T lymphocytes correlates with the encephalitogenicity of the cells and their ability to infiltrate the CNS during clinical disease. Little or no HPSE expression was observed in CD8+ T cells, macrophages, or astrocytes during disease progression. These findings support the notion that inducible HPSE expression during EAE is regulated at the level of transcription in CNS-infiltrating CD4+ T cells. The study was then extended to explore the mechanisms that regulate inducible HPSE transcription in human primary T lymphocytes under a physiological setting, i.e., activation via CD3 and CD28. HPSE mRNA expression was shown to be induced in naive T cells in response to activation with anti-CD3 and anti-CD28 and, furthermore, that this correlated with increased HPSE activity. These findings confirm that inducible HPSE expression in human primary T cells is also regulated at the transcriptional level. Functional analysis of the HPSE promoter in primary human T cells was used to locate a 280-bp minimal inducible region and also suggested that the transcription factor, early growth response gene 1 (EGR1) is an important regulator of inducible HPSE transcription. This was strongly supported by the key observation that EGR1 siRNA inhibited inducible HPSE mRNA expression in activated primary human T cells. These data for the first time describe the kinetics and cell type-specific inducible expression of HPSE in EAE and demonstrate that it is controlled at the level of transcription and also provides definitive evidence that EGR1 is a crucial regulatory factor.
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Rat immunization and culture of T blast cells
For induction of antigen specific CD4+ T cells, rats were immunized with 25 µg myelin basic protein (MBP) in Complete Freund’s adjuvant (CFA) containing 4 mg/ml Mycobacterium butyricum (Bacto, Sydney, Australia), as described previously [23
]. At 7-9 days postimmunization, draining lymph nodes were harvested and processed into a single-cell suspension in RPMI containing 10% FCS, 5 x 10–5 M 2-Mercaptoethanol (Gibco) and 2 mM glutamine. Freshly isolated lymph node cell suspensions were cultured for 3 days with antigen presenting cells (APC) at a ratio of 1:50 if irradiated thymocytes were used as APC or 1: 0.02 if mitomycin C treated splenocytes were used as APC and 25 µg/ml MBP. Antigen-presenting cells were then removed using a density gradient 1.077 (Ficoll-Paque Research Grade, Pharmacia Biotech, Perstorp, Sweden), as described by the manufacturer, and the T blast cells were cultured in IL-2-containing medium. After 7–14 days of culture in IL-2 medium, cells reverted back to resting cells, at which time they were restimulated with antigen and antigen-presenting cells as described above. Alternatively, these cells were activated with either Con A 4 µg/ml or by plastic immobilized anti-CD3 (BD PharMingen, San Jose, CA, USA).
Actively induced EAE and immunohistochemistry
EAE was induced in Lewis rats by subcutaneous immunization with 25 µg MBP in CFA. At preclinical (7 days), clinical (11 days), and recovery (14 days) stages of EAE, lumbar spinal cords were taken and fixed in 4% paraformaldehyde. After antigen retrieval with citrate buffer pH 6.0, 4-µm serial sections were used for immunohistochemical staining. HPSE was detected using a rabbit anti-rat HPSE [32
], macrophages and activated microglia using a biotinylated mouse anti-rat ED1 (Serotec, Oxford, United Kingdom), astrocytes with a rabbit anti-GFAP (Chemicon, Boronia, Australia), and CD8+ T cells with a mouse biotinlyated anti-rat CD8 (Serotec, Oxford, United Kingdom). Nonspecific binding was assessed using an antibody to the human testis specific four-transmembrane molecule TETM4 [33
]. Antibody binding was detected using the DAKO LSAB-2 System HRP (Carpinteria, CA, USA). Sections were counterstained with Gill's III hematoxylin. Cytospin slides of CD4+ or CD8+ T cells were stained for rat HPSE using an IHC AEC Kit (InnoGenex, San Ramon, CA, USA) as per the manufacturer’s instructions with a rabbit anti-rat Hpse antibody [32
] with nonspecific binding assessed as described above.
Isolation and culture of primary human T lymphocytes
Human primary T lymphocytes were isolated by negative selection using a modification of a protocol used to isolate human NK cells [34
]. Whole peripheral blood was collected from healthy human volunteers into heparinized tubes by informed consent under studies approved by the human ethics committee of the Australian National University. Ficoll-Hypaque (Amersham Biosciences Pharmacia, Uppsala, Sweden) was overlaid by blood diluted at a ratio of 1:2 in 0.5% BSA/PBS, and centrifuged at 600 g at room temperature for 20 min. Peripheral blood mononuclear cells (PBMC) were harvested and washed twice in 0.5% BSA/PBS. PBMC and freshly isolated red blood cells (RBC) suspended in 0.5% BSA/PBS were then combined at a ratio of 1:100 in a total volume of 45 x 106 PBMC/ ml. To this suspension was added 50 µl RosetteSep human T cell enrichment antibody cocktail (Stem Cell Technologies, Vancouver, Canada). The suspension was incubated at room temperature for 20 min, diluted 1:2 in 0.5% BSA/PBS, overlaid on RosetteSep DM-L (Stem Cell Technologies, Vancouver, Canada) and then centrifuged at 600 g for 20 min without braking. T cells were harvested from the interface and subjected to two rounds of washes in 0.5% BSA/PBS. T cells were then resuspended at a concentration of 1.25 x 106 cells/ ml in 10% FCS/RPMI supplemented with 0.1% PSN and 2 mM glutamine and seeded in a Linbro 12-well plate (ICN Biomedical, Aurora, OH). In activation experiments, the media were supplemented with anti-CD28 (gift of Dr. Sudha Rao, The John Curtin School of Medical Research, Canberra, Australia) at a ratio of 1:5,000, and the cells were cultured in plates precoated with 10 µg/ml anti-CD3 clone HIT3a (BD Biosciences PharMingen, San Jose, CA, USA). The identity of all T lymphocytes used in subsequent experiments was confirmed by flow cytometry to be greater than 97% CD3 positive.
Heparanase activity assay
HPSE activity assays were carried out on 0.75 x 106 T lymphocytes that were either unstimulated or stimulated with anti-CD3 and anti-CD28 for 24, 48, 72, or 96 h, as described previously [35
]. Briefly, cells were centrifuged at 1200 g for 10 min, and media were carefully removed. Cell pellets were then frozen on dry ice and stored at – 70°C. Cell pellets were resuspended in 50 µl 0.1% Triton-X100 and underwent three rounds of freeze and thaw cycles. An aliquot of each sample was then incubated with 90 pmol of 3H-labeled HS (see below for details) in 0.05 M sodium acetate buffer, pH 5.1, containing 0.1 mg/ml BSA, 5 mM N-acetylmannosamine (NAM) and 0.15% Triton X-100 (Sigma, St Louis, MO, USA) in a total volume of 40 µl. Triplicates were carried out for each sample, as well as for blank measurements. Samples were incubated overnight at 37°C. The reaction was stopped by placing samples immediately onto dry ice and adding 40 µl heparin (10 mg/ml). Next, 340 µl of cetypyridinium chloride (CPC) (Sigma) was added to samples, followed by vortexing and incubation at 37°C for 30 min. Samples were centrifuged prior to addition of 100 µl of supernatant along with scintillation fluid to scintillation vials, then assayed in a Packard Tri-Carb 1500 Liquid Scintillation Analyzer (Parkard Instruments, Meriden, CT, USA). The production method and physiochemical properties of the 3H-labeled HS have been described in detail previously [35
]. Briefly, purified porcine mucosal HS (number-average molecular mass of 18.5 kDa) was partially de-N-acetylated and re-N-acetylated with 3H-acetic anhydride (Amersham International, Sydney, Australia) to a specific radioactivity of 1650 to 2000 c.p.m./pmol. The labeling procedure did not introduce any new or altered cleavage sites and was validated as a suitable natural substrate for the detection of HPSE activity, as described previously [35
].
RNA extraction and real-time quantitative RT-PCR
Total RNA was extracted from 4 x 106 human T lymphocytes that were unstimulated or stimulated with anti-CD3 and anti-CD28 for 24, 48, 72, or 96 h or 1 x 106 rat CD4+ lymphocytes that were unstimulated or stimulated with cross-linked anti-CD3 or Concanavalin A (Con A) (Sigma), using TriReagent (Molecular Research Center Inc., Cincinnati, OH, USA), as described by the manufacturer. cDNA synthesis was performed on 1 µg of total RNA using Superscript III reverse transcriptase, as per the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). SYBR Green (Qiagen, Hilden, Germany) real-time PCR reactions for amplification of human HPSE, human EGR1, or the housekeeper gene ubiquitin-conjugating enzyme E2D 2 (UBC) were performed using an ABI PRISM 7700 sequence detector (PerkinElmer, Shelton, CO, USA), as described previously [32
]. Amplification of rat Hpse or Ubc genes were performed using oligonucleotides, MHRT1 (5'-GTTCCTGTCCATCACCATCGA-3') and RHRT2 (5'-CGTGGAGAGCCCAGGAAGGT-3'), or RATUBC1 (5'-TGAAGAGAATCACAAGGAACTGA-3') and RATUBC2 (5'-CGACAGGACCTGCTGAACACTGT-3'), respectively. A dissociation curve was performed after each experiment to confirm a single product was amplified. Relative mRNA expression was calculated using the 2(-Delta Delta C(T)) method [36
]. Relative changes in expression were analyzed for statistical significance by using a two-tailed Student’s t test.
Cell lysates and Western blot analysis
Western blot analysis was performed on 5.25 x 106 primary T lymphocytes that were either unstimulated or stimulated with 10 µg/ml plastic immobilized anti-CD3 and 1:5,000 anti-CD28 for 24, 48, 72, or 96 h. Western blot analysis was used to detect HPSE in cell lysates using a rabbit polyclonal antibody specific for both 50 and 65 kDa human HPSE (gift of Dr. Craig Freeman, John Curtin School of Medical Research, Canberra, Australia) following the protocol described previously [37
]. Chemiluminescence was detected using ECL Western blotting detection reagents, as described by the manufacturer (Amersham Biosciences, UK).
Plasmid constructs, transient transfections and optimization of luciferase reporter assays
Luciferase reporter constructs containing fragments of the human HPSE promoter; pXP-1/1300bp, pXP-1/520bp, pXP-1/280bp, pXP-1/120bp, pXP-1/280bpMUT, and pCR3.1/EGR1 have been described previously [32
,38
]. Functional analysis of the human HPSE promoter was investigated in primary human T cells following the optimization of a transient transfection protocol as follows. The capacity to efficiently transfect human primary T cells was first assessed by transfection with an expression construct encoding for enhanced green fluorescence protein (EGFP). Transient transfection of primary T lymphocytes was carried out using a Human T cell Nucleofector Kit and a Nucleofector (Amaxa, Cologne, Germany). Initially, 3 x 106 T cells resuspended in 100 µl of T cell nucleofector solution were added to 3 µg pEGFP-N1 (Clontech BD Biosciences, San Jose, CA, USA) and transferred to a nucleofector cuvette. Cells were electroporated using program U-14 (nonstimulated cells) or T-23 (stimulated cells), then transferred in prewarmed media to a 12-well plate and incubated at 37°C. Cells were assessed under a UV light microscope (Olympus, Melville, NY, USA) for green fluorescence at several intervals after transfection. Expression of EGFP was detected as early as 2 h after transfection of cells and was sustained for several days. Cells assessed at 24 h post-transfection, routinely expressed green fluorescence at greater than 50% of the cell population (data not shown).
The optimal time of luciferase protein expression in primary human T lymphocytes using the Nucleofector transfection system was determined by cotransfecting cells previously activated for 5 days with anti-CD3 and anti-CD28, with the firefly construct pXP-1/280bp (previously been shown to have the greatest activity in Jurkat T cells [32 ]), along with a Renilla construct, pRLTK. 3 x 106 cells resuspended in 100 µl of T cell nucleofector solution were added to 3 µg firefly reporter construct and 1 µg Renilla reporter construct and transfected as described above. Cells were harvested at 4, 6, or 9 h after transfection and firefly and Renilla luciferase assayed using a Dual-Glo Luciferase assay system (Promega, Madison, WI, USA). Plates were read on a Reporter Microplate Luminometer (Turner Biosystems, Sunnyvale, CA, USA). Background luminescence, as measured on an equal number of untransfected T cells, was subtracted from all samples. Both firefly and Renilla luciferase activity were determined to be greatest at 4 h after transfection, with a greater than 50% reduction in luminescence by 9 h posttransfection (data not shown). This early expression and rapid degradation of luciferase is consistent with the direct delivery of the DNA to the nucleus of the cell as achieved by the Nucleofector technology and the variable stability of luciferase protein in primary cells. Consequently, all transfections of primary T lymphocytes cells were carried on freshly isolated human T lymphocytes that were either unstimulated or previously activated with anti-CD3 and anti-CD28 for 5 days and luciferase assays performed 4 h after transfection. Transfection of an aliquot of T cells with pEGFP-N1 was performed as a positive control for each reporter assay. For the EGR1 overexpression experiments, cells were transfected as described above, with a constant amount of total DNA used, that is, when decreasing amounts of pCR3.1/EGR-1 were transfected, the balance was made up by cotransfection with the backbone construct, pCR3.1. Overexpression studies and mutagenesis effects were analyzed for statistical significance by using a two-tailed Student’s t test.
EGR1 RNAi knockdown of HPSE expression in primary human T-lymphocytes
Human primary T lymphocytes were freshly isolated and transiently transfected with EGR1 siRNA (sc-29303) or negative control siRNA (sc-37007) (Santa Cruz Biotechnology, CA, USA) using a human T cell Nucleofector Kit and a Nucleofector (Amaxa) as described above but with the following modifications. 3 x 106 T cells resuspended in 100 µl of T cell nucleofector solution were added to 1.5 µg EGR1 siRNA or control siRNA and electroporated using program T-23. Cells were then rested for 12 h before stimulation or not with anti-CD3 and anti-CD28 as described above. Cells were harvested at 24 and 48 h after stimulation and HPSE or EGR1 mRNA levels were determined by real-time RT-PCR as described above. Results were analyzed for statistical significance by using a two-tailed Student’s t test.
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Figure 1. HPSE is expressed in activated rat CD4+ T cells. (A) Hpse is expressed in rat primary activated CD4+, but not CD8+ T cells. Cytospin slides of purified rat primary CD4+ or CD8+ T cells were stained with an anti-rat Hpse specific rabbit polyclonal antibody. No staining of CD4+ or CD8+ cells was observed with a control rabbit antibody (data not shown). (B) HPSE mRNA levels are up-regulated in rat MBP specific CD4+ T lymphocytes upon stimulation with Con A as measured using real time quantitative RT-PCR. Total RNA was extracted from 1 x 10 6 rat MBP specific CD4+ T lymphocytes either non-stimulated (NS) or stimulated for 18 h, 30 h, 3, 4, and 7 days. First-strand cDNA was synthesized from 1 µg of total RNA and then subjected to real-time RT-PCR analysis. Expression relative to nonstimulated cells was calculated using the 2(-Delta Delta C(T)) Method. The data represent the mean ± SE of 2 independent experiments. p NS indicates the P value is not significant.
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Figure 2. HPSE is expressed in encephalitogenic cells. (A) Rat MBP-specific CD4+ T lymphocytes were activated with antigen-presenting cells (APC) and 25 µg/ml of MBP for 3 days. APC were then removed using a density gradient and CD4+ T cells cultured in IL-2 containing medium. Cells were harvested after 1 day (representing encephalitogenic T cells) or 4 days (representing nonencephalitogenic T cells) culture in IL-2 containing medium and total RNA extracted from 1 x 106 cells. First-strand cDNA was synthesized from 1 µg of total RNA and then subjected to real-time PCR analysis as described in Fig. 5
. The data represent the mean ± SE of two independent experiments. (B) Rat MBP-specific CD4+ T lymphocytes were activated as described in (A), and an aliquot of cells harvested after 1 day (representing encephalitogenic T cells) or 7 days (representing nonencephalitogenic T cells) culture in IL-2-containing medium. Cytospins were performed to transfer the cells to slides. The slides were stained with either an anti-rat Hpse specific rabbit polyclonal antibody or a control rabbit antibody. Inset highlights a selected field showing cell surface staining of HPSE indicated as by arrowheads.
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Figure 3. HPSE is expressed by CNS infiltrating CD4+ T cells in EAE. Lumbar spinal cords were taken from Lewis rats on days 7 (preclinical EAE), 11 (clinical EAE), and 14 (recovery from EAE) after immunization with MBP in CFA. Four-micron serial sections of formalin-fixed and paraffin-embedded specimen were used for immunohistochemical staining after antigen retrieval. The staining for heparanase, ED1 for macrophages/activated microglia, GFAP for astrocytes, and an isotype control antibody are shown.
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15, 000 copies of HPSE mRNA per 1 µg total RNA (data not shown). HPSE mRNA increased 2.2-fold compared with unstimulated cells after 48 h of activation, with a peak in expression of 2.7-fold occurring at 72 h after activation, changes that were highly statistically significant (Fig. 4A
). This expression is sustained at 96 h. T cells stimulated as described above were also analyzed for HPSE protein expression using Western blot analysis. HPSE protein representing the processed 50-kDa active form of the enzyme was induced at 72 h and sustained to 96 h (Fig. 4B)
. At 72 and 96 h, a faint band corresponding to the 65-kDa pro-form of HPSE could also be detected. To determine whether the induction of HPSE mRNA and protein expression in primary T lymphocytes corresponded to an induction in enzyme activity, HPSE activity assays were carried out on cell lysates collected from primary T lymphocytes activated with anti-CD3 and anti-CD28 for 24, 48, 72, and 96 h. HPSE enzymatic activity was low in unstimulated cells and gradually increased over 96 h, at which time it was four-fold greater than that of unstimulated cells (Fig. 4C)
. Overall, the kinetics of inducible mRNA and protein HPSE expression correlated well with enzyme activity. However, it should be noted that at 96 h, HPSE activity is slightly higher than expected based on the determined HPSE protein levels by Western blot analysis. We have recently shown that HPSE is readily secreted from T lymphocytes following activation and that it can accumulate in the culture media and bind back to the cell surface (M. D. Hulett, unpublished observations). It is most likely that the higher level of HPSE activity when compared with the protein level at the 96-h time point is due to additional HPSE activity from the enzyme binding back to the surface of cells, a source of HPSE that was not seen in our Western blot analysis that only detected intracellular protein.
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Figure 4. HPSE mRNA and protein expression in human primary T lymphocytes. (A) HPSE mRNA expression is up-regulated in activated primary T lymphocytes as measured by real-time RT-PCR. Total RNA was extracted from 4 x 106 cells that were either nonstimulated (NS) or stimulated with plastic immobilized anti-CD3 and soluble anti-CD28 for 24, 48, 72, and 96 h. NS at 0 h is shown, and NS samples at each time point displayed similar low levels of HPSE mRNA (data not shown). First-strand cDNA was synthesized from 1 µg of total RNA and then subjected to real-time PCR analysis. HPSE mRNA expression is expressed relative to nonstimulated cells and was calculated using the 2(-Delta Delta C(T)) method [37
]. The results represent the mean ± SE of 2 independent experiments. (B) Western blot analysis using a human HPSE antibody was carried out on 5.25 x 106 human T lymphocytes either resting (NS) or activated with anti-CD3 and anti-CD28 for 24, 48, 72, or 96 h. NS at 0 h is shown, and NS samples at each time point displayed similar low levels of HPSE protein (data not shown). Purified HPSE was included as a positive control (+). A 50-kDa HPSE-specific band is indicated. An additional 65-kDa band representing the proform of HPSE is also present in the 72- and 96-h samples and is indicated by an arrowhead. The position of the 53-kDa molecular weight marker is shown. A Coomassie Blue stained gel is shown as a loading control. The image is representative of two independent experiments. (C) HPSE enzymatic activity is correlated with HPSE expression. HPSE enzymatic activity was assayed on 0.75 x 106 human T lymphocytes that were either unstimulated (NS) or stimulated with anti-CD3 and anti-CD28 for 24, 48, 72, or 96 h. NS at 0 h is shown and NS samples at each time point displayed similar low levels of HPSE activity (data not shown). Each sample was measured in triplicate. Two independent experiments were carried out and the data represent the mean ± SE from 1 representative experiment. p NS indicates the P value not significant.
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Figure 5. EGR1 regulates inducible HPSE transcription in human primary T lymphocytes. (A) Basal and inducible HPSE promoter activity was assessed using HPSE promoter reporter constructs containing varying lengths of the HPSE promoter in the luciferase vector pXP1 (pXP-1/1300bp, pXP-1/520bp, pXP-1/280bp, pXP-1/120bp). Each construct together with a Renilla control construct were transfected into primary T cells, which were nonstimulated (NS) or had been previously stimulated with anti-CD3 and anti-CD28 for 5 days. At 4 h after transfection, cells were harvested, and the luciferase activity was measured for firefly and Renilla luciferase using the Dual-Glo luciferase assay system. Luciferase ratio was calculated as firefly expression relative to Renilla expression. The data represent triplicate transfections and the error bars represent the mean ± SE. p NS indicates the Pvalue is not significant. (B) Wild-type construct pXP-1/280bp or pXP-1/280bpMUT (containing a transversion mutant of the core sequence of the putative EGR1 binding site in the 280-bp fragment of the HPSE promoter) together with a Renilla control construct were transfected into human primary T cells and that were either nonstimulated (NS) or previously stimulated with anti-CD3 and anti-CD28 for 5 days. Luciferase assays were performed as described in Fig. 3
. Each transfection was performed in triplicate. The data are representative of the mean ± SE. p NS indicates the P value is not significant. (C) Cotransfection of an EGR1 expression construct with pXP-1/280bp increases HPSE promoter activity. pXP-1/280bp was transfected along with increasing amounts of EGR1 expression construct (pCR3.1/EGR1) and the appropriate amount of empty backbone pCR3.1 into human primary T cells, which were previously nonstimulated and luciferase assays were performed as described in Fig. 3
. Each transfection was carried out in triplicate. The data are representative of the mean fold change in promoter activity relative to transfection with the empty vectors pXP-1 and pCR3.1 +/– SE. (D) EGR1 siRNA inhibits inducible EGR1 mRNA expression in activated primary human T cells. Human primary T cells were freshly isolated and transiently transfected with EGR1 siRNA or a negative control siRNA and nonstimulated (NS) or stimulated with anti-CD3 and anti-CD28 for 24 or 48 h. First-strand cDNA was synthesized from 1 µg total RNA isolated from cell samples and subjected to real-time PCR analysis. EGR1 mRNA expression relative to nonstimulated cells (NS) was calculated using the 2(-Delta Delta C(T)) method. The data represent the mean ± SE. (E) EGR1 siRNA inhibits inducible HPSE mRNA expression in activated primary human T cells. Human primary T cells were freshly isolated, transiently transfected with EGR1 siRNA or a negative control siRNA and nonstimulated (NS) or stimulated with anti-CD3 and anti-CD28 for 24 or 48 h, before being analyzed as above for HPSE mRNA by real-time PCR. The data represent the mean ± SE.
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To directly determine the role of EGR1 in the control of inducible HPSE mRNA expression in T cells, the effect on HPSE expression of down-regulating EGR1 levels using EGR1 siRNA was assessed. Primary human T cells were transfected with EGR1 siRNA or a negative control siRNA and stimulated with anti-CD3 and anti-CD28. The EGR1 siRNA greatly reduced EGR1 mRNA levels in T cells that had been stimulated for 24 h to a relative expression of 2.1 times that of unstimulated cells when compared with the control siRNA-treated cells that showed a 26-fold greater expression than unstimulated cells, clearly demonstrating that this approach was effective in reducing EGR1 expression (Fig. 5D) . It should be noted that at 48-h poststimulation EGR1 mRNA levels had declined markedly, which is consistent with the kinetics of EGR1 expression following activation of T cells [32 ]. Significantly, the knockdown of EGR1 expression also markedly reduced the levels of inducible HPSE mRNA expression in T cells that had been stimulated for 24 or 48 h to a relative expression of 1.2 and 1.4 times that of unstimulated cells when compared with the negative control siRNA-treated cells that showed a 1.8- and 2.9-fold greater expression than unstimulated cells, respectively (Fig. 5E) . These data indicate that EGR1 is an important regulator of inducible HPSE expression in primary human T cells.
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The results obtained with both rat antigen-specific CD4+ T cells and human primary T cells suggest that HPSE mRNA expression in lymphocytes does not peak until 3 or 4 days post activation. Previous studies on the induction of HPSE have all been carried out in rat antigen specific CD4+ T cells and were restricted to secreted HPSE activity assays. Initial studies reported induced activity by ECM bound and soluble antigen at 48 and 72 h postactivation [8 ]. A later study reported a gradual increase in HPSE activity over 6 h, as well as secreted activity being present at 24 and 72 h postactivation [25 ]. Bartlett et al. (26) showed that HPSE activity was induced in human primary lymphocytes in response to PMA activation for 24 h. Fridman et al. (25) also reported that HPSE activity could be detected as early as 5 min postactivation, and, furthermore, this early induction of secreted HPSE activity could not be inhibited by incubation of cells with a protein synthesis inhibitor. This supported the notion that HPSE existed preformed in an intracellular compartment in memory T cells and was released as an early event in response to the activation of cells. Interestingly, Fridman and colleagues also observed a gradual decline in HPSE activity in the presence of a protein synthesis inhibitor from 1.5 h postactivation, with a 50% reduction in HPSE activity relative to control cells occurring at the longest time point assayed, 6 h. This suggests a protein synthesis-dependent source of HPSE was released at these later time points. The relative contribution of secreted prestored vs. secreted synthesized HPSE remains unknown. The findings of Fridman et al. together with those described herein of the late induction of HPSE expression and activity support the notion that there are two phases of inducible HPSE activity in memory T cells; early release of prestored HPSE and a late-phase induction of HPSE expression and activity. It might be expected that naive T cells would be restricted to the late-phase induction of expression. This is supported by the absence of HPSE protein expression in freshly isolated human mononuclear cells [10 ] and the finding of low HPSE protein expression in cell lysates from resting primary T lymphocytes as reported in this study.
Several studies with both T and B lymphocytes have shown that EGR1 is induced in response to antigen cross-linking of the TCR and BCR, respectively [44 ]. A more recent study with T cells indicated that EGR1 could be induced by both MHC-dependent and MHC-independent mechanisms, i.e., in the absence of exogenous antigen [45 ]. Furthermore, TCR agonists and partial agonists induce EGR1 expression that corresponds with the strength of the agonist [44 ]. In this study, we present the first definitive evidence that EGR1 regulates inducible HPSE expression in primary T lymphocytes. This was directly demonstrated in that EGR1 siRNA inhibited inducible HPSE mRNA expression in activated primary human T lymphocytes. These data are supported by transactivation and promoter mutagenesis experiments described herein, together with our previous observations using chromatin immunoprecipitation that EGR1 interacts directly with the HPSE promoter in vivo [38 ]. In addition, we have also shown that HPSE and EGR1 expression colocalizes to infiltrating mononuclear cells in EAE [32 ].
It should be noted that the transcription factors Sp1 and Ets-1 have been implicated in regulating HPSE transcription in tumor cells [33 ,38 ,46 ,47 ]. Furthermore, we have previously demonstrated that Sp1 also binds to the HPSE promoter in unstimulated and stimulated Jurkat T cells [32 ]. It is known that EGR1 mediates transactivation events through a number of different mechanisms that are dependent on the interaction with other transcription factors, coactivators or repressors such as CBP, p300, and NAB1 or NAB2 and also its phosphorylated state [48 ]. In addition, EGR1/SP1 modules have been described in promoters of several other genes [49 ,50 ]. To further clarify the mechanisms by which EGR1 regulates HPSE expression, it would therefore be interesting to study the expression of factors such as Sp1, Ets-1, CBP, p300, NAB1, and NAB2, particularly the kinetics of the recruitment of these factors to the HPSE promoter, together with the phosphorylation status of EGR1 under these experimental conditions. It is also of interest to note that we have recently shown that EGR1 plays a key role in regulating HPSE gene transcription in tumor cells [38 ]. Significantly, HPSE transcription is inducible in tumor cells in response to activation signals, suggesting that EGR1 has a universal role in regulating inducible HPSE expression in a number of cell types.
EAE is a T cell-mediated autoimmune disease of rodents that is studied as a model of the human disease, multiple sclerosis. The effector cells involved in the development of disease are CD4+ Th1 cells specific for myelin antigens, such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or S100β [40 ]. Critical to the development of the disease is an inflammatory process. This involves the migration of activated CD4+ and CD8+ T cells and monocytes across the subendothelial basement membrane and their entry and subsequent accumulation in the central nervous system (CNS). In addition, cytokines and chemokines secreted by T cells and macrophages, as well as the activation of resident CNS cells, such as microglia and astrocytes [51 ] and reactivation of effector T cells [52 ] have been shown to be critical for the development of an intense inflammatory response necessary for mediating disease. Early studies proposed that the promigratory function of Hpse would indicate a role for Hpse in the entry of activated effector T cells into the CNS. This was supported by the correlation of Hpse activity in antigen-specific T cells with the ability of the cells to cause disease [8 ] and that certain sulfated polysaccharides, which inhibit disease, also inhibit Hpse activity [20 21 22 ]. These later studies also showed a total absence of inflammatory infiltrate in treated rats [20 ,22 ]. The findings reported in this study that Hpse is expressed in CD4+ T cells crossing blood vessels and infiltrating the perivascular areas and meninges in spinal cord sections from rats during clinical disease but not the recovery stages provides significant support to a role for Hpse in the passage of these cells across the blood-brain barrier (BBB). Significantly, the in vitro studies described herein also provide support for this role, as Hpse is preferentially expressed in encephalitogenic but not in nonencephalitogenic T cells. Furthermore, Hpse expression in MBP-specific CD4+ T cells in vitro showed a peak in expression at 4 days postactivation. In the context of the model of actively induced EAE described herein, this expression correlates well with the time of maximal accumulation of T lymphocytes in the CNS during disease [41 ].
While much of the focus on Hpse in the context of EAE has been on T lymphocytes, it is plausible that HPSE may be expressed during EAE in other cell types that contribute to the inflammatory response, such as macrophages, astrocytes, and endothelial cells. Furthermore, it may be involved in the regulation of cytokine and chemokine release. Previous work in rat primary astrocytes indicated that Hpse mRNA is induced in response to LPS activation, and this induction is inhibited by the heparin-like molecule, RG-13577 [20 ]. The findings described herein by examination of a rat model of actively induced EAE suggest that it is the infiltrating CD4+ T cells that express Hpse, with no expression detected in macrophages, activated microglia, astrocytes, or CD8+ T cells. However, it should be noted that Hpse expression was detected in some endothelial cells in the CNS of rats with active EAE, as we have described previously [32 ]. The expression of Hpse in endothelial cells suggests that the enzyme may also contribute to the progression of disease in a non-T lymphocyte manner. Indeed, it has recently been shown that in DTH reactivation in the mouse ear that endothelial cell-derived heparanase appears to play an important role in DTH-associated inflammation [24 ]. It will clearly be of interest to determine the relative contribution of heparanase derived from CD4+ T cells and endothelial cells to the progression of EAE.
The lack of Hpse induction in CD8+ T cells is intriguing and suggests that the efficient passage of these cells into the CNS may be dependent upon other Hpse-expressing cell types, namely CD4+ T cells and/or endothelial cells. Of relevance to this observation is that the majority of inflammatory cells in the CNS lesions are recruited mononuclear cells that enter the CNS after the initial wave of encephalitogenic T cells. The high level of Hpse expression in activated CD4+ T cells as opposed to CD8+ T cells suggests that Hpse transcription is regulated differently in these cell types. We have shown that CD8+ T cells are capable of expressing Egr1 mRNA (A. M. de Mestre, unpublished observations), suggesting that another level of control exists in these cells. The mechanism of differential expression of HPSE in T lymphocytes is currently being investigated. It is also interesting to note that the encephalitogenic rat MBP-specific CD4+ T cells showed some surface staining for Hpse. Macrophages and dendritic cells have recently been shown to express Hpse on their cell surfaces upon activation that promotes efficient ECM degradation [12 ,13 ]. The detection of Hpse on the surface of activated T cells suggests these cells may also use this mechanism to promote efficient cell invasion.
In conclusion, this study presents evidence that HPSE expression is regulated at the level of transcription in primary T lymphocytes, and furthermore that the induction of HPSE mRNA expression results in corresponding HPSE enzymatic activity. It is also demonstrated that the transcription factor, EGR1, is critical in regulating inducible HPSE expression in human primary T cells. In addition, this study provides the first direct evidence that Hpse is expressed in infiltrating T cells during active disease in EAE. This study provides a platform for further investigation to better understand the role of HPSE and also EGR1 in EAE and autoimmunity. Finally, the observation that inducible HPSE expression in T cells is controlled at the level of transcription by EGR1, identifies a potential novel target for inhibiting HPSE expression and therefore HPSE function in T cell-mediated inflammatory disease. In support of this notion, EGR1 has been proven a successful target in the treatment of animal models of tumor progression and vascular proliferative disorders [53 54 55 ].
Received May 16, 2007; revised June 22, 2007; accepted July 2, 2007.
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