Inflammation, Vol. 22, No. 6, 1998
ACTIVATED T-LYMPHOCYTES EXPRESS OCCLUDIN, A COMPONENT OF TIGHT JUNCTIONS J. S. ALEXANDER,1 T. DAYTON,2 C. DAVIS,1 STEPHEN HILL,1 T. HALLER JACKSON,1 OREST BLASCHUK,3 MATTHEW SYMONDS,3 NAOTSUKA OKAYAMA,1 CHRIS G. KEVIL,1 F. STEPHEN LAROUX,1 S. M. BERNEY,2 and D. KIMPEL,2 1501 Kings Highway Louisiana State University Medical Center Shreveport, Louisiana, 71130 1 Department of Molecular and Cellular Physiology and 2 Department of Rheumatology 3 Department of Urology, Royal Victoria Hospital 687 Pine Ave West Montreal, Canada, H3AIA1
Abstract—T-lymphocytes routinely traffic from the lymphoid and vascular compartments to the tissues during immune surveillance and inflammatory responses. This egress occurs without compromising endothelial barrier, which is maintained by tight junctions (zonula occludens). We report that T-lymphocytes up-regulate the expression of occludin, a major component of the tight junction in response to stimulation with phorbol ester (PMA) + calcium ionophore, CD3 antibody or T-cell receptor (TCR) antibody. Only activated T-lymphocytes express occludin; this adhesion molecule is nearly absent in resting T-lymphocytes. By immunofluorescence, occludin is seen in lymphocyte aggregates, but does not appear to mediate aggregation since only 50% of the cells in these clusters express occludin. Occludin is expressed between 8 and 24 h following stimulation, and persists for at least 48 h. These data indicate that activated T cells produce occludin which may regulate lymphocyte adhesion and trafficking.
INTRODUCTION T-Lymphocytes control many of the immune surveillance and regulatory functions of the immune system, and also mediate several forms of acute and chronic inflammatory reactions including graft rejection and autoimmunity (1). During 573 0360-3997/98/1200-0573$l5.00/0 c 1998 Plenum Publishing Corporation
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normal functioning, T-lymphocytes migrate from the vascular lumen to the tissue as part of immune surveillance, and eventually return to the lymphatic compartment (2). To perform these functions, T-lymphocytes must normally cross several cellular and tissue barriers which maintain and regulate a perm-selective barrier to the exchange of soluble materials (3-5). The mechanisms that allow leukocytes to gain access to these different compartments are not completely understood. Most leukocytes (like lymphocytes) traverse the endothelial monolayer, at endothelial adherens and occludens junctions, which contain cadherins and occludin (1,2,7,9). Since the solute barrier is preserved during migration, migrating leukocytes must somehow preserve endothelial junctions during extravasation, but the molecules and mechanisms regulating these events remain poorly understood. Several classes of proteins are known to mediate the adhesion of leukocytes to the endothelium. Lymphocytes are known to adhere to endothelial monolayers through several lymphocyte adhesion molecules which include LFA-1, VLA-4, a1bl, a4b1, aLb2, aMb1 and endothelial adhesion molecules including PECAM-1, ICAM-1 and VCAM-1 (5-9). While there is some evidence that indicates that these molecules may also modulate migration, roles for these molecules in transmigration remain controversial. Since leukocytes pass across both endothelial and epithelial monolayers at cell-cell junctions, many groups have recently focused on the roles of endothelial junction molecules in transmigration. Cadherins are calcium-dependent adhesion molecules that mediate normal cell-cell adhesion in both endothelial and epithelial cells (10,11). Some recent studies indicate that some transformed types of lymphocytes (Jurkat) can express cadherins (12). This finding immediately suggests that lymphocyte cadherins may permit lymphocytes to pass across adherens junctions by transiently sealing with junction proteins as it passes through the barrier (13). However, less is known about how the tight junction (zonula occludens) participates in this event. Occludin is a novel 59 kD important transmembrane component of the tight junction, or zonula occludens, which forms at least part of the solute barrier in epithelial and endothelial monolayers (14). Occludin has only been described in endothelial and epithelial cells, however, since lymphocyte cadherins have been recently described, (12), we examined whether, and under what conditions lymphocytes might also express occludin. We observed that occludin was expressed at low levels in unstimulated T-lymphocytes, and that several types of pharmacological and physiological stimuli dramatically increased its expression. T lymphocyte occludin occurs as the same 59 kD protein reported in endothelial and epithelial cells, and is found in approximately 50% of stimulated CD4+ and CD8+ T-lymphocytes. Occludin was nearly absent in nonstimulated T-lymphocytes, and is also absent in B-lymphocytes. Structurally, occludin in stimulated T-lymphocytes is concentrated at cell-cell borders of aggregated lym-
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phocytes. The maximal expression of occludin occurs 24-48 h after stimulation with phorbol ester + calcium ionophore, anti-CD3 and anti-T-cell receptor antibodies. These data suggest that occludin may fulfill several important adhesive and homing functions in activated T-lymphocytes that are related to antigen specific stimulation via the TCR/CD3 complex.
MATERIALS AND METHODS Isolation of Lymphocytes. Rat mesenteric, inguinal, brachial, and popliteal lymph nodes were pooled from anesthetized animals. Lymphocytes were isolated by macerating tissue against a 70 um nylon mesh screen (Falcon) and washing the tissue homogenate with medium conssiting of RPMI with 5% fetal calf serum. These isolated cells were collected by centrifugation at 300 x g. Cells were re-suspended in 1 ml of medium, and a 20 ul aliquot counted in a Coulter XL-1 cells counter (Coulter instruments, Hialeah, Florida). Antibodies. Several antibodies were used in these studies for western blotting, immunostaining, fluorescence activated cell sorting and cell stimulation. The occludin antibody was obtained from Zymed laboratories, (San Francisco, California). The CD45R antibody, is specific for rat B-lymphocytes (15), and was obtained from Pharmingen corp., (Missisauga, Ontario, Canada). The CD3, ab T-cell receptor ('TCR'), CD4 and CD8 antibodies were also obtained from Pharmingen. Stimulation of Lymphocytes. Isolated lymphocytes were stimulated by three methods: 1) stimulation for 4, 8, 24, and 48 h with a combination of calcium ionophore (A23187, Sigma) 250 ng/ml and phorbol 12-myristate 13-acetate (PMA, Sigma) 30 ng/ml; 2) stimulation for 48 h with 60 ug of anti-rat CD3 antibody (Pharmingen, clone G4.18), 3) stimulation for 48 h with 60 ug of T-cell receptor antibody (Pharmingen). To perform antibody stimulation, 60 ug of antibody was bound to the surface of a T-75 flask, cells added, and at the appropriate time points, cells harvested, and a 10 ul aliquot counted by Coulter counting. Aliquots of 107 cells were snap frozen at -80°C for Western blotting. Fluorescence Activated Cell Sorting of Lymphocytes. After 48 h of stimulation with PMA/ionomycin, cells were harvested and re-suspended at 5 x 107 cells/ml with a 1:200 dilution of primary (anti-CD4, anti CD8 or anti CD45R). After incubating for 30 min at 4°C, cells were washed twice and separated by fluorescence activated cell sorting (FACS) into CD4+/CD8- and CD8+/CD4- populations, or CD45R+ and CD45R- populations on a FACS Vantage flow cytometer (Becton Dickinson). Aliquots of 2 x 106 cells of each type were pelleted at 15000 x g, (1 min), and snap frozen for Western blotting. Immunofluorescence. Stimulated and non-stimulated cells were affixed to coverslips by coating the coverslips with a 1:1 mixture of ovalbumin and glycerol. Twenty ul of this solution was placed on 12-mm coverslips, and at the appropriate time points, 30 ul of cell suspension placed on this emulsion. Coverslips were then fixed for 30 min in 4°C methanol, followed by 1 min in acetone at 25°C and air-dried. Coverslips were incubated with 1:500 rabbit anti-occludin antibody (Zymed) for 1 h in 0.1 milk powder in Dulbecco's PBS, washed 3x in this buffer, reacted with 1:100 goat rhodamine labeled anti-rabbit IgG (Jackson Labs), washed 3x in this buffer and mounted onto slides in 10 ul of glycerol: PBS with 0.1% p-phenylenediamine (1:1). Cells were photographed at 100 x on T-MAX professional film. Immunoblotting. Controls containing 2 x 106 cell aliquots of non-stimulated and stimulated cells were snap frozen, and later lysed in 60 ul of sample buffer (1% SDS, 0.5 M Tris, pH 8, 1% 2-mercaptoethanol with 1 ug/ml aprotinin, leupeptin and 1 mM phenylmethylsulfonyl fluoride) at
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65°C and 15 ul (equivalent to 500,000 cells) loaded onto 7.5% SDS/PAGE gels. Proteins were electrophoresed at 150 V, 35 mA and transferred to nitrocellulose. After staining blots with Ponceau dye to verify transfer, blots were blocked with 5% milk powder and incubated for 1 h with 1:1000 anti-occludin (Zymed) in 0.1% milk powder, washed 3 x (5' each wash) and developed with 1:2000 goat anti-rabbit alkaline phosphatase (Sigma, St. Louis, Missouri), washed 3 x (5' each wash) and reacted with NBT/BCIP chromogen to visualize the proteins. Quantitation of immunoblots was carried out by capturing the image on a flat bed scanner and analysis of pixel density using Image Pro Plus™ software. Northern Blotting. We performed northern blotting according to our previously described method (16). Total RNA was prepared from approximately 2 x 106 cells using the Rneasy total RNA kit (Qiagen, Chatsworth, California). A total of 10 ug of total RNA was applied to each lane for gel electrophoresis. 15 ul RNA samples were mixed with 2.5 u1 of sterile 6x loading buffer [0.25% w/v bromophenol blue, 0.25% w/v xylene cyanol, 30% w/v glycerol, 1.2% SDS, 60 mM sodium phosphate (pH 6.8)]. Samples were denatured at 75°C for 5 min and immediately loaded on to a 1.2% agarose gel. The 1.2% agarose gel and running buffer were made with Ix TBE containing 1 ug/ml of ethidium bromide (10 ug/ml stock). The gel was electrophoresed 100 V for 75 min. Zeta-Probe GT membrane (Bio-Rad) and transfer filter papers were equilibrated in Ix TBE for 10 min. The gel was then transferred to the nylon membrane using the Genie electrophoretic blotter (Idea Scientific, Minneapolis, Minnesota) at 12 V for 90 min. The membrane was then U.V. cross-linked for 5 min and incubated in 10 ml of pre-hybridization solution (0.25M Na 2 HPO 4 (pH 7.2) and 7% SDS) for 30 min at 65°C. Northern Probes. GAPDH (ATCC, Rockville, Maryland) and occludin probes were labeled to a specific activity of at least 1 x 109 CPM/ug using the Ready-to go Random Primer Oligo Kit (Pharmacia, Piscataway, New Jersey). Probes were purified using Sephadex G-50 spin columns (Pharmacia, Piscataway, New Jersey). Denatured probes were added to the prehybridization solution and hybridized at 55-60°C for 4-8 h. After hybridization, membranes were washed successively with vigorous agitation at room temperature for 15 min in each of the following solutions: 2x SSC/0.1% SDS, 0.5x SSC/0.1% SDS, 0.lx SSC/0.1% SDS. Membranes were then wrapped in cling-film (saran wrap) and exposed to film for 12-24 h at -70°C.
RESULTS Occludin Is Expressed by Lymphocytes Stimulated with Phorbol Myristate Acetate (PMA) and Calcium Ionophore (A23187). Figure la shows western blotting for the presence of occludin in lymphocytes stimulated with PMA and A23187 for time points up to 48 h. While freshly isolated lymphocyte preparations do not express occludin, after PMA + A23187 stimulation, these lymphocytes express occludin at 24 and 48 h. A low level of expression is present in unstimulated cells. There appears to be a doublet of occludin expressed, similar to the pattern observed in endothelial and epithelial cells in culture. Occludin Message Is Increased in PMA + A23187 Stimulated Lymphocytes. In Figure 1b, Northern blotting for the presence of occludin mRNA shows the conventional 1.8 kB message for occludin as well as the 'novel' 5.0 kB message present in stimulated, but not control lymphocytes. No occludin message is detectable in non-stimulated cells, but both the 1.8 and 5.0 kB message are
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Fig. 1. Occludin is Expressed by Lymphocytes Stimulated with Phorbol Myristate Acetate (PMA) and Calcium Ionophore (A23187). Figure la shows western blotting for the presence of occludin in lymphocytes stimulated with PMA nad A23187 for time points up to 48 h. While freshly isolated lymphocyte preparations do not express occludin, PMA + A23187 stimulated lymphocytes express occludin at 24 and 48 h. There appears to be a "doublet" of occludin expressed in these cells, similar to the pattern observed in endothelial and epithelial cells in culture. Figure 1b. Occludin message is increased in PMA + A23187 stimulated lymphocytes. Northern blotting for the presence of the conventional 1.8 kB message for occludin as well as for a "novel" 5.0 kB message for occludin indicates that neither messages for occludin is detectable in non-stimulated cells, but both are appear at 12 h. Both the 1.8 and 5.0 messages are present at 24 and 48 h, however, the 5.0 kB message is decreased by 48 h.
detected after 12 h. Both the 1.8 and 5.0 kB messages are dramatically increased at 24 and 48 h, however, it appears that the 5.0 kB message is beginning to decrease by 48 h. This indicates that the appearance of message for occludin precedes the appearance of occludin protein by several hours, suggesting that the protein detected by western blotting is newly synthesized instead of being released from a storage site. Occludin Expression in PMA + A23187 Stimulated Lymphocytes Is Heterogeneous. Figure 2 shows a low level of fluorescence associated with unstimulated lymphocytes (Figure 2A), whereas PMA + A23187 stimulated lymphocytes show a bright and heterogeneous expression of occludin (Figure 2B). Approximately 50% of the cells in these aggregates appear to express high levels of occludin. Lymphocytes which are stimulated with PMA + A23187 typically form clusters of >30 cells. It appears unlikely that this aggregation is mediated by occludin since not all cells within these clusters express occludin. Stimulation of Cells via T-Cell receptor/CDS Complex Increases Occludin
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Fig. 2. Occludin expression in PMA + A23187 Stimulated Lymphocytes is heterogeneous. Immunostaining of lymphocytes under control conditions shows a low level of fluorescence associated with un-stimulated lymphocytes (a), whereas PMA + A23187 stimulated lymphocytes (b) show a bright, heterogeneous expression of occludin. Approximately 50% of the cells in these aggregates appear to express high levels of occludin. Lymphocytes stimulated with PMA + A23187 typically form clusters of >50 cells. It appears unlikely that this aggregation is mediated solely by occludin since not all cells within these clusters express occludin (see arrows. Figure 2b).
Expression in Lymphocytes. Lymphocytes were also stimulated using anti-CD3 antibody or anti T-cell receptor antibody. Figure 3 shows Western blots following stimulation by each of these antibodies and control antibodies, each at 4 ug/ml, in comparison to pharmacologic stimulation (PMA + A23187). Expression of occludin is increased by stimulation through TCR or CD3 antibodies, although the level of expression is much lower than that with PMA+ A23187 stimulation. Lymphocytes stimulated with control antibodies were to unstimulated cells in the absence of occludin expression. Both CD4+ and CD8+ Lymphocytes Express Occludin. In order to further distinguish which T-lymphocyte subsets expressed occludin following stimulation, we used fluorescence activated cell sorting to separate CD4+ and CD8+ lymphocytes after 48 h with and without PMA + A23187 stimulation (Figure 4a). Equal numbers of each cell type were prepared for Western blotting. Figure 4a shows that both CD4+ and CD8+ lymphocytes expressed occludin. The CD4+ cells expressed 200% more occludin than CD8+ cells, based on densitometry. The non-stimulated CD4+ or CD8+ cells failed to express occludin. Purified T-Lymphocytes Up-Regulate Occludin Expression in Response to PMA + A23187. In order to determine whether B or T lymphocytes were
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Fig. 3. TCR-Complex Specific Stimuli Increase Occludin Expression in Lymphocytes. Figure 3 shows that in comparison to stimulation with PMA + A23187, T-Iymphocytes exposed to 4 ug/ml anti-CD3 antibody, or 4 ug/ml anti T-cell receptor (TCR) increase expression of occludin. By comparison, non-stimulated cells show dramatically less occludin expression. Controls for antibody stimulation were performed using isotype matched antibodies at the same concentration.
expressing occludin, we stimulated cells isolated from lymph nodes, (a mixture of B and T lymphocytes), for 48 h with PMA + A23187 and then performed fluorescence activated cell sorting (FACS) using a rat B-cell specific antibody, CD45R (15). After separating B-lymphocytes (CD45R+) from T-lymphocytes (CD45R-) we performed western blotting for occludin. In Figure 4b, we observed that at 48 h, T-lymphocytes (CD45R-), expressed occludin. Some of the unstimulated T-lymphocytes also produced a small amount of occludin, although the expression is much greater in PMA + A23187 stimulated cells.
DISCUSSION Lymphocyte trafficing to areas of inflammation is mediated by several classes of adhesion molecules in the immunoglobulin, proteoglycan and lectin superfamilies (5,6,9). Cadherins are important components of endothelial and epithelial adherens junctions (10,11,17) and have been found in lymphocytes (12). These molecules, and their interactions with integrins may contribute to the transepithelial and transendothelial movement of lymphocytes (12). While
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Fig. 4. (a) Both CD4+ and CD8+ Lymphocytes Express Occludin. In order to further distinguish which T-lymphocytes express occludin following stimulation with PMA + A23187, we used fluorescence activated cell sorting to separate CD4+ and CD8+ lymphocytes following PMA + A23187 stimulation (48 h) and after 48 h without stimulation. Figure 4a shows that both CD4+ and CD8+ lymphocytes expressed occludin, with CD4+ cells expressing 220% more occludin than CD8+ cells, based on densitometry. After sorting, we found that non-stimulated CD4+ or CD8+ cells failed to show expression of occludin. (b) T-Lymphocytes Up-Regulate Occludin Expression in Response to PMA + A23187. We observed that at 48 h stimulated T-lymphocytes (CD45R-) expressed occludin. Some of the unstimulated T-lymphocytes also produce occludin after 48 h in culture, although the effect is much more remarkable in PMA + A23187 stimulated cells. In CD45R+, (B-lymphocytes) did not express occludin in this set of experiments,
cadherins and occludin are generally characterized as homotypically associating, only one study has shown the presence of cadherins in lymphocytes (12), which might permit cadherin homotypic interactions to mediate adhesion and migration. However, there are no data in the literature which demonstrate a leukocyte form of occludin which could participate in these types of interactions. In many tissues, tight junctions (zonula occludens) constitute significant barriers to soluble materials in the vascular compartment (11). Junctions may as well create a barrier to the exchange of leukocytes (1,18) however, the interactions of leukocytes with tight junctions have not been extensively examined. We report that stimulation of lymphocytes with either phorbol myristate acetate + calcium ionophore or anit-CD 3 antibody or anti-T cell receptor (TCR) antibody induces the expression of occludin, the main component of endothelial and epithelial tight jucntions. Kago, et al. recently demonstrated that CD28 on aggregated T-lymphocytes induces the formation of tight junction-like cell contacts, and is also associated with actin polymerization and accumulation of rho GTPase
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at these contacts (19). Therefore, it is likely that a similar type of activation may occur in response to the CD3 and T-cell receptor antibody challenge. Our observation that occludin present in activated T-lymphocytes has dramatic implications for the normal functioning of T-lymphocytes. T-lymphocytes normally circulate between lymph nodes, the spleen and target tissues within the body and consequently traverse several junctional barriers in the endothelium and epithelium. Therefore endothelial and epithelial junctions must be transiently disrupted to allow leukocytes to cross these junctions. Lymphocyte expression of occludin, (a tight junction component) might facilitate both the binding and extravasation of T-lymphocytes across endothelial and epithelial layers, and importantly might allow migration of these leukocytes without an interruption of the normal solute barrier. Occludin in T-lymphocytes may also target T-lymphocytes to occludin rich junctions that might provide additional specificity or enhanced anchoring in lymphocyte homing. The time course experiment (Figure 1) shows expression of occludin mRNA after 12 h of stimulation, with maximal expression at 24 and 48 h. Likewise protein expression is dramatically increased at the 24 and 48 h time points. Therefore occludin expression in lymphocytes must involve de novo mRNA and protein synthesis. Similar results are also observed for RT-PCR of occludin in stimulated cells (data not shown). Stimulated lymphocytes express both the typical 1.8 kB message for occludin, as well as the 'novel' 5.4 kB occludin message (Figure Ib). Additionally, we noted that at 24-48 h, non-stimualted T-lymphocytes (Figure 4b, CD45R-, control) will also express low levels of occludin, due perhaps to non-specific activation produced by the isolation and culture protocol. However, occludin expression is relatively low compared to stimulated cells, and is not expressed at high levels at early time points (see Figure la at 0, 4 and 8h). In conclusion, the expression of occludin by activated T-lymphocytes may be an important, novel adhesive or migratory mechanism that could regulate lymphocyte-endothelial interactions involved in normal lymphocyte functions and in lymphocyte mediated pathologies. Subsequent studies indicate that purified B-lymphocytes may constitutively express and possibly up-regulate occludin expression. This observation is the focus of future studies.
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