ORIGINAL ARTICLE
Inhibition of Contact Sensitivity by Farnesylthiosalicylic Acid-Amide, a Potential Rap1 Inhibitor Adam Mor1,2, Roni Haklai2, Ofer Ben-Moshe2, Yoseph A. Mekori3,4 and Yoel Kloog2,4 We hypothesized that Ras proximate 1 (Rap1) functions as an additional target for farnesylthiosalicylic acid (FTS) or its derivatives, and that the inhibition of Rap1 in lymphocytes by these agents may represent a method for treating inflammatory disorders. Indeed, we found that FTS-amide (FTS-A) was able to inhibit the elicitation phase of delayed cutaneous hypersensitivity in vivo. This effect was associated with the inhibition of Rap1 more than with the inhibition of Harvey rat sarcoma viral oncogene (Ras). Moreover, FTS-A inhibited Rap1 and contact sensitivity far better than FTS. We suggest that FTS-A may serve as a possible therapeutic tool in contact sensitivity in particular and T-cell-mediated inflammation in general. Journal of Investigative Dermatology (2011) 131, 2040–2048; doi:10.1038/jid.2011.152; published online 30 June 2011
INTRODUCTION Lymphocytes are the major cellular component of the adaptive immune response. Normal function of lymphocytes depends on several small guanosine nucleotide-binding proteins (also known as small G proteins). This superfamily of proteins consists of over 50 members that cycle between an inactive guanosine diphosphate bound state and an active guanosine triphosphate (GTP) bound state. These proteins are involved in a variety of signal transduction pathways that regulate lymphocyte growth, trafficking, migration, and apoptosis (Scheele et al., 2007). Among the most studied are Harvey rat sarcoma viral oncogene (Ras), Rheb, Rho, Rac, and Ras proximate 1 (Rap1; Scheele et al., 2007). Both Rap1 and Ras are highly expressed in T lymphocytes. Rap1 was first identified as an antagonist for Ras. Active Rap1 can bind but not activate Raf-1, and thereby may sequester Raf-1 from the Ras/extracellular signal-regulated kinase pathway (Mor et al., 2007a). Another pathway through which Rap1 can antagonize Ras function in T cells is the suppression of Ras-dependent reactive oxygen species. 1
Department of Medicine, New York University School of Medicine, New York, New York, USA; 2Department of Neurobiology, The George Weise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel and 3 Department of Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 4
Yoel Kloog and Yoseph A. Mekori contributed equally to this work.
Correspondence: Yoel Kloog, Department of Neurobiology, The George Weise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. E-mail:
[email protected] Abbreviations: FTS, farnesylthiosalicylate; FTS-A, farnesylthiosalicylateamide; FTS-MOM, farnesylthiosalicylate-methoxymethylester; GFP, green fluorescent protein; GTP, guanosine triphosphate; PLD1, phospholipase D1; Rap1, ras proximate 1; Ras, Harvey rat sarcoma viral oncogene; TNF, tumor necrosis factor Received 30 July 2010; revised 10 April 2011; accepted 16 April 2011; published online 30 June 2011
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Finally, p38 activation by IL-1, characterized as Ras dependent, is antagonized by Rap1. In contrast, in other pathways, Rap1 and Ras synergize. For example, TCR stimulation results in activation of both Rap1 and Ras. These findings are an example of the Rap1–Ras complexity and the concept of Ras antagonism by Rap1 remains controversial (Dillon et al., 2005; Mor et al., 2007a). Growth control, protein trafficking, and polarity are some of the processes in which Rap1 has been implicated. The best-characterized and most prominent function of Rap1 is to promote lymphocyte function-associated antigen-1-mediated adhesion. Lymphocyte function-associated antigen-1 activation by Rap1 is a critical step for lymphocytes homing to peripheral lymph nodes and migrating into inflamed tissues (Dillon et al., 2005). Inhibition of Rap1 abrogates lymphocyte function-associated antigen-1-mediated adhesion to antigenpresenting cells and IL-2 production (Mor et al., 2007a). In some patients with leukocyte adhesion deficiency III, a defect in Rap1 GTP loading is responsible for the profound defect in lymphocyte adhesiveness (Abram and Lowell, 2009; Mor et al., 2009), which highlighting the critical role of Rap1 in host defense. Small G protein-dependent signaling requires both GTP binding and inner cell membrane anchorage. These proteins are attached to lipid membranes through a lipid moiety, such as farnesyl, prenyl, or gernylgernyl. Approaches to inhibit Ras include the use of Ras inhibitors farnesylation (farnesyltransferase inhibitors; Zhu et al., 2003) and the use of S-transfarnesylthiosalicylic acid (FTS), a synthetic compound that resembles the carboxyl-terminal farnesylcysteine of Ras (Pando et al., 2010). Cells readily take up FTS, and once inside the cell, it specifically disrupts the association of active forms of Ras with the inner surface of the cell membranes (Goldberg et al., 2008). FTS reduced the amount of Ras in a dose-dependent manner (Weisz et al., 1999; George et al., & 2011 The Society for Investigative Dermatology
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RESULTS FTS derivatives inhibit Rap1 in Jurkat T cells
We inquired whether FTS and its derivatives are able to inhibit GTP loading of Rap1. Quiescent Jurkat T cells were treated
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overnight with FTS, FTS-A, and FTS-methoxymethylester (MOM), each at a concentration of 50 mM. Cells were collected, and the quantity of GTP-Rap1 was determined by pull-down assay. Compared with the untreated cells (control), the amount of GTP-Rap1 decreased in all conditions (Figure 1a). However, both FTS-A and FTS-MOM were superior to FTS in their ability to inhibit Rap1 activation (Figure 1a). Rap1 is activated in T cells as a result of crosslinking the antigen receptor. We wanted to study the effect of FTS, and its derivatives, on Rap1 activation in stimulated T cells. Cells were treated with FTS, and its derivatives overnight, and subsequently stimulated with anti-CD3 antibodies for 10 minutes. As shown in Figure 1b, all derivatives were able to inhibit Rap1 activation in stimulated T cells. FTS lowered the amount of GTP-Rap1 by 32%, whereas FTS-A and FTSMOM lowered it by 60 and 53%, respectively (Figure 1c). Thus, FTS and its derivatives are able to inhibit Rap1 activation in T cells stimulated through the antigen receptor. However, it seems that FTS-A and FTS-MOM were more potent Rap1 inhibitors compared with FTS.
←GTP-Rap1 ←Total Rap1 ←GTP-Rap1 ←Total Rap1
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2004). The selectivity of FTS to active Ras may explain the relatively non-toxic nature of this compound. When FTS was given to patients with malignancies in clinical trials, adverse effects were uncommon (http://clinicaltrials.gov). FTS has been shown to have beneficial effects not only in animal models of proliferative diseases but also in experimental autoimmune neuritis (Kafri et al., 2005), MRL/lpr lupus prone mice (Katzav et al., 2001), and myocarditis (Pando et al., 2010). Although FTS interferes with activated Ras membrane anchorage, it can also affect directly or indirectly other small G-binding proteins (Goldberg and Kloog, 2006). For example, FTS shifts the balance between Rac1 and Rho activation (Zundelevich et al., 2007). FTS is also an inhibitor of isoprenylcysteine carboxyl methyltransferase (Marom et al., 1995). A potential Ras inhibitor should prevent both farnesylation and geranylgeranylation of Ras. FTS, with its 15-carbon farnesyl moiety, was found to inhibit both farnesylation and geranylgeranylation of Ras (Elad et al., 1999; Jansen et al., 1999). Farnesyltransferase inhibitors, on the other hand, inhibit only Ras farnesylation, thus limiting its potential clinical use as a therapeutic agent. We hypothesized that Rap1 functions as an additional target for FTS or its derivatives, and that the inhibition of Rap1 in lymphocytes may represent a, previously unreported, method for treating inflammatory skin disorders. Surprisingly, we found that FTS was able to inhibit the elicitation phase of delayed cutaneous hypersensitivity in vivo. This effect was associated with inhibition of Rap1 more than with the inhibition of Ras. Moreover, FTS-amide (FTS-A) inhibited Rap1 and contact sensitivity far better than FTS. Contact sensitivity is the immunological response to an allergen, which may lead to allergic contact dermatitis. The underlying pathophysiology of this disorder is exposure to an antigen that activates T cells, thereby promoting the release of inflammatory mediators, resulting in a delayed phase cutaneous reaction. The established pharmacological agents available to treat this disorder are limited. Options include glucocorticosteroids and antihistamines. It is of great value to develop a new therapeutic tool in contact sensitivity for several reasons. First, current agents lack suitable effectiveness. Second, considerable side effects of the existing drugs often limit treatment, especially in long-term or recurrent management. In addition, a, to our knowledge, previously unreported agent may be potentially administered before an exposure known to trigger contact sensitivity in order to prevent the inflammatory response. To develop a more effective management, research should identify new pathways for intervention. Cutaneous exposure to antigens triggers a diversity of intracellular signaling cascades—many of which are Rap1 dependent. Thus, FTS-A might be of great significance in serving as a therapeutic agent in contact sensitivity, mediated through the inhibition of Rap1.
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Figure 1. FTS derivatives inhibit Rap1 in Jurkat T cells. Jurkat T cells were treated overnight with FTS, FTS-A, and FTS-MOM (50 mM). GTP-loaded Rap1 was determined by pull-down assay (a). Jurkat T cells were treated with FTS and its derivatives overnight (50 mM), and subsequently stimulated with anti-CD3 antibodies for 10 minutes. GTP-Rap1 was determined by pull-down assay (b). Quantification of the same experiments (n ¼ 4, mean±s.e.m) is shown in c. *Pp0.05; relative to control. FTS, farnesylthiosalicylic acid; FTS-A, FTS-amide; FTS-MOM, FTS-methoxymethylester; GTP, guanosine triphosphate; Rap1, ras proximate 1.
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Figure 2. FTS derivatives inhibit T-cell adhesion. Jurkat T cells were treated overnight with FTS or its derivatives (50 mM) as indicated. Cells were plated on ICAM-1-coated wells for 20 minutes followed by serial washings. A plate reader was used to evaluate the percentage of cells that remained in the wells (n ¼ 4, mean±s.e.m). *Pp0.05; relative to control. FTS, farnesylthiosalicylic acid; FTS-A, FTS-amide; FTS-MOM, FTS-methoxymethylester; NT, not treated.
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As Rap1 is critical for T-cell adhesion, we tested the ability of FTS and its derivatives to inhibit adhesion of T cells to ICAM-1-coated plates. T cells were treated overnight with FTS or its derivatives. Cells were plated on ICAM-1-coated wells for 20 minutes followed by serial washings. A plate reader was used to evaluate the percentage of cells that remained in the wells. Although 40% of stimulated T cells attached to the ICAM-1-coated wells, this number dropped by half in wells that were treated with the derivatives (Figure 2). No statistically significant difference could be found among the three agents. Thus, FTS, and its derivatives, inhibit both Rap1 GTP loading and Rap1-dependent T-cell adhesion.
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FTS-A inhibits Rap1 activation at the plasma membrane
We have previously reported that only the pool of Rap1 at the plasma membrane becomes GTP bound on lymphocyte activation (Mor et al., 2009). We wanted to study whether Rap1 inhibition by FTS-A is indeed restricted to that 2042 Journal of Investigative Dermatology (2011), Volume 131
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Out of the three agents (FTS, FTS-A, and FTS-MOM), we chose to focus on FTS-A mainly because of its superiority in terms of Rap1 inhibition (Figure 1c). First, we treated T cells with various concentration of FTS-A for 72 hours. As shown in Figure 3a, with higher dose, the number of viable cells decreased, as assessed by trypan blue exclusion. Notably, with higher dose of FTS-A, the amount of GTP-Rap1 among the viable cells (dead cells were excluded based on specific density centrifugation) further diminished, indicating a dose–response relationship (Figure 3b). We limited the concentration of FTS-A in this experiment to 50 mmol l1, as further inhibition was accompanied by the loss of cell viability. Interestingly, only high concentration of FTS-A inhibited adhesion to ICAM-1-coated plates (Figure 3c).
l
FTS-A inhibition of Rap1 is dose dependent
Figure 3. FTS-A inhibition of Rap1 is dose dependent. Jurkat T cells were treated with various concentration of FTS-A (as indicated) for 72 hours. Viable cells were assessed by trypan blue exclusion (n ¼ 3, mean±s.e.m) (a). Jurkat T cells were treated with different concentrations of FTS-A (as indicated) and the amount of GTP-Rap1 was evaluated by pull-down assay (n ¼ 3, mean±s.e.m) (b). T cells were treated with different concentrations of FTS-A and adhesion to ICAM-1-coated wells was studied as described earlier (n ¼ 4, mean±s.e.m; c). *Pp0.05; relative to control. FTS, farnesylthiosalicylic acid; FTS-A, FTS-amide; NT, not treated; Rap1, ras proximate 1.
compartment. Jurkat T cells, expressing green fluorescent protein (GFP)-tagged Rap1, were subjected to various agents (Figure 4a), whereas the localization of Rap1 was recorded by confocal microscopy. As previously reported, knocking down phospholipase D 1 (PLD1) resulted in inhibition of Rap1 and its association with the plasma membrane (Figure 4a). FTS-A
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Figure 4. FTS-A inhibits Rap1 activation at the plasma membrane. Jurkat T cells, expressing GTP-tagged Rap1, were subjected to various agents (as indicated) and the localization of Rap1 was recorded by confocal microscopy (a). Jurkat T cells expressing the probe for activated Rap1, GFP-RBDRALGDS were stimulated through the antigen receptor, and the localization of the probe was recorded by confocal microscopy with and without pretreatment with FTS-A at 50 mM (b). Bars ¼ 5 mm (a, b). FTS-A, farnesylthiosalicylic acid-amide; GFP, green fluorescent protein; PLD-1, phospholipase-1; Rap1, ras proximate 1; shRNA, short hairpin RNA.
treatment for up to 72 hours did not change the localization of overexpressed Rap1 (Figure 4a). Rap1, similar to K-Ras, is associated with the plasma membrane through farnesylation that functions in conjunction with an adjacent polybasic sequence. Bryostatin-1, a protein kinase C agonist, induced a rapid translocation of K-Ras from the plasma membrane to intracellular membranes (Bivona et al., 2006). Nonetheless, we found that a combined treatment with bryostatin-1 and FTS-A did not change the localization of Rap-1 (Figure 4a). Thus, FTS-A does not change the bulk localization of Rap1. The effects of FTS are rather specific to the active GTP-bound forms of Ras proteins (Marom et al., 1995). To investigate the possible effect of FTS-A on localization of the active Rap1, we took advantage of the probe for activated Rap1, GFP-RBDRALGDS (Bivona and Philips, 2005). Consistent with earlier reports (Bivona et al., 2004), when the cells were stimulated through the antigen receptor, the probe translocated to the plasma membrane, suggesting that Rap1 is activated at that compartment (Figure 4b). In cells that were pretreated by FTS-A, the activation of Rap1 at the plasma membrane was blocked (Figure 4b), although Rap1 remained associated with the plasma membrane (Figure 4a). Thus, the pool of Rap1, which is inhibited by FTS-A, is found at the plasma membrane. FTS-A inhibits contact sensitivity reaction in animal model
Next, the ability of FTS-A to inhibit Rap1-dependent T-cell adhesion in vivo was investigated by using contact sensitivity
(Claman and Jaffee, 1983) as a model system. Animals were treated orally with two different concentrations of FTS-A (50 and 100 mg kg1). Topical betamethasone (0.05%) served as positive control. As shown in Figure 5a, high concentration FTS-A was able to inhibit ear swelling. This effect was more prominent after 48 hours (Figure 5a). When treatment was introduced only during the challenge phase (days 5–6), ear swelling was also attenuated. Treatment during the sensitization phase (days 0–2) did not prevent ear swelling (Figure 5b), suggesting that FTS-A primarily blocked lymphocyte recruitment to the site of foreign antigen encounter. To characterize the inflammatory response at the cellular level, mice were treated as above, and at 48 hours latter, the epicutaneous challenged ears were cut and analyzed. Heavy mononuclear cell infiltrate characterizes the positive control group (Figure 5c) in correlation with the macroscopic appearance of the ear. Both FTS-A and topical betamethasone reduced the cellular infiltrate (Figure 5c). There was no significant difference between the ability of FTS-A or betamethasone to reduce the infiltrate. The majority of these cells were T lymphocytes (CD3 positive; Figure 5d). Few mast cells and B cells were detected in all conditions (Figure 5d). Quantification of the cellular infiltrate in this experiment revealed a significantly reduced number of T cells in the treatment group (Figure 5e). These cells expressed Rap1 (Figure 5d). Finally, we tested whether FTS-A could affect the systemic inflammatory response triggered by exposure to DNFB (2,4-Dinitro-l-fluorobenzene). Although not statistically significant, serum levels of tumor necrosis factor (TNF)-a were attenuated when treated with FTS-A (Figure 5f). Rap1 inhibition by FTS-A is not associated with Ras inhibition
As FTS was shown to inhibit Ras, we inquired whether the mechanism of Rap1 inhibition by FTS derivatives is indirect and mediated by Ras inhibition. We compared the inhibitory profile of FTS and its derivatives on both Ras GTP loading and Rap1 GTP loading in Panc-1 cells (Figure 6a). As previously reported, FTS-MOM was a weak Ras inhibitor, whereas FTS was comparable with FTS-A. In contrast, FTS-MOM was by far the stronger Rap1 inhibitor, whereas FTS was modest (Figure 6a). Thus, the extent of Ras and Rap1 inhibition by FTS and its derivatives is not identical. Furthermore, when we knocked down N-Ras and K-Ras by short hairpin RNA (shRNA) in Jurkat T cells, the inhibitory effect of FTS-A on Rap1 was not interrupted, suggesting again that Rap1 inhibition is probably not Ras dependent (Figure 6b). DISCUSSION In this study, we demonstrated that, rather surprisingly, Rap1 activation is inhibited by FTS and its derivatives. We found that FTS-A is by far a better inhibitor of Rap1 than FTS (Figure 6a and b), suggesting that the effect of FTS-A on contact sensitivity (Figure 5a) is through the inhibition of Rap1. We also showed that Rap1-mediated adhesion of lymphocytes was blocked by FTS-A. As there is considerable cross-talk between small G proteins, a major concern is whether the inhibitory effect of FTS-A on Rap1 is indeed direct or www.jidonline.org 2043
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alternatively mediated through linkage to Ras. In this study, we presented evidence that does not support such a Ras–Rap1 linkage. The effect of FTS-A on Rap1 activation was unchanged when Ras was knocked down, suggesting that Ras was not required for both Rap1 activation and for FTS-AControl
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mediated inhibition of Rap1 activation (Figure 6b). Similar results were obtained in experiments with dominant-negative Ras (data not shown). FTS-MOM was a strong Rap1 inhibitor (Figure 6a), whereas it had only a minor inhibitory effect on Ras (Figure 6b; Goldberg et al., 2009). These results
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agent for delayed cutaneous hypersensitivity syndrome, a common skin disorder with a median prevalence of 21% (Thyssen et al., 2007; Carlsen et al., 2008). To our knowledge, this is previously unreported. Our study is not free of weaknesses and limitations. We can not be certain that our model for contact allergic dermatitis in laboratory animals accurately represents cutaneous immunology in humans, in whom the effect on dendritic cells might be different (Bergstresser, 1989). In addition, our therapeutic interventions were not able to
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demonstrate a distinct profile of inhibition for these two small G-binding proteins: relatively high selectivity of FTS toward Ras and of FTS-MOM toward Rap1. Although FTS-A inhibited both Ras and Rap1, FTS-MOM was more selective toward Rap1; we chose to use FTS-A for the contact sensitivity model system. We did so because FTS-A showed the highest antiRap1 activity. Rap1 is the key factor in the development of delayed cutaneous hypersensitivity syndrome, and it is likely that FTS-A affects mostly Rap1 under these circumstances. Our results suggest that FTS-A is a potential new therapeutic
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Figure 6. Rap1 inhibition by FTS-A is not associated with Ras inhibition. Panc-1 cells were treated with different FTS derivatives (50 mM) and pull-down assay for GTP loaded Rap1 and Ras were recorded (a). N-Ras and K-Ras were knocked down in Jurkat T cells (with shRNA) and the localization of activated Rap1 was evaluated (b). Bars ¼ 5 mm (b). FTS, farnesylthiosalicylic acid; FTS-A, FTS-amide; FTS-MOM, FTS-methoxymethylester; GFP, green fluorescent protein; GTP, guanosine triphosphate; Rap1, ras proximate 1; shRNA, short hairpin RNA.
Figure 5. FTS-A inhibits contact sensitivity reaction in animal models. Animals were treated orally with two different concentrations of FTS-A (50 and 100 mg kg1) or with daily topical application of betamethasone (0.05%) and ear swelling was evaluated after induction of contact sensitivity (a). Animals were treated with FTS-A during the challenge phase (days 5–6) or during the sensitization phase (days 0–2), and ear swelling was studied (b). Macroscopic and histological assessment of the ears of mice after treatments as indicated above; sections were stained with H&E and imaged at 10 and 20 magnification (c). Immunohistochemical evaluations of tissue sections with anti-CD3, B220, Rap1, and toluidine blue were imaged at 40 magnification (d). Quantification of the number of CD3-positive cells per high-power field views (e) were recorded. Mice were bled at 48 hours after challenge and serum level of TNF-a was documented by ELISA (f). Results are shown as mean±s.e.m (n ¼ 5–10 in each group). *Pp0.05; relative to control. Bars ¼ 100 mm (c) and 50 mm (d). FTS-A, farnesylthiosalicylic acid-amide; H&E, hematoxylin and eosin; Rap1, ras proximate 1; TNF-a, tumor necrosis factor.
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demonstrate a significant effect on serum TNF-a levels. Thus, the cytokine levels should have been studied by tissue mRNA quantification. In a review of the current literature, several investigators published observations that further disrupt the association between Ras and Rap1 (Mor et al., 2007a). Mst1 is a kinase that is found downstream of both Ras and Rap1 (Mor et al., 2007a). In one study, cells treated with FTS did not show any divergence in Mst1 activation, whereas Ras was significantly inhibited (Pando et al., 2009). Others showed that cellular adhesion is one of the Ras-independent Rap1 functions (Carey et al., 2000; Dillon et al., 2005; Mor et al., 2009). Thus, it seems that the inhibitory effect of FTS on these small G proteins is not interrelated (Pando et al., 2009). Previous work was carried out in an effort to find new FTS derivatives that might improve the anti-cancer effect of the drug (Goldberg et al., 2009). It was shown that specific modifications of the FTS carboxyl group by esterification (e.g., FTS-MOM) or amidation (e.g., FTS-A) yielded compounds with improved growth inhibition activity (Goldberg et al., 2009) and with improved tissue penetration by its increased lipophilicity (Goldberg et al., 2009). Of all derivatives, FTS-A seems to be the most effective. FTS-A exhibits good bioavailability after oral administration, has an excellent safety profile, and may thus be considered for clinical trials (Barkan et al., 2006; Haklai et al., 2008). In this study, we showed that FTS-A was the most potent Rap1 inhibitor in T cells stimulated by the antigen receptor. Interestingly, the FTS geranylgeranyl analog did not prove to be a stronger Rap1 inhibitor (data not shown). In this study, we showed that the development of contact sensitivity reaction to DNFB in animal models was strongly inhibited by FTS-A. Contact sensitivity to DNFB has been extensively studied in mice. It requires both effective immune sensitization following cutaneous exposure to chemical haptens- and antigen-specific elicitation (Zimber et al., 1982; Bryce et al., 2004). We showed that treatment with FTS-A during the second exposure to the antigen was sufficient to block ear swelling. It is, therefore, suggested that the drug can block the recruitment of the primed T cells to the inflamed area. Thus, we suggest that FTS-A might be a promising candidate to treat patients with inflammatory diseases, in which T-cell adhesion and recruitment has a major role. MATERIALS AND METHODS General reagents The 5-carboxyuorescein and Opti-MEMI were purchased from Invitrogen Corporation/Molecular Probes (Carlsbad, CA). Bryostatin-1 was provided by Sigma-Aldrich (St Louis, MO). FTS was synthesized, as previously described (Haklai et al., 2008), and was stored in chloroform, which was evaporated under a stream of nitrogen immediately before use. FTS-MOM and FTS-A were provided by the Concordia Pharmaceuticals (Fort Lauderdale, FL).
Antibodies and DNA constructs Mouse anti-human CD3 (Ancell, Bayport, MN) was used for T-cell activation. 2046 Journal of Investigative Dermatology (2011), Volume 131
Anti-Rap1antibody was purchased from BD Biosciences (San Jose, CA). Monoclonal anti-Ras antibody (Ras10) was purchased from Calbiochem (San Diego, CA). For immunohistochemistry, tissue sections were stained with anti-B220 (BD Biosciences), antiCD3, and anti-Rap1 antibodies (Abcam, Cambridge, MA). GFP-Rap1WT, pcDNA-Rap1WT, pcDNA-Rap1N17, GFP-RalGDSRBD, shRNA-RFP/H1-PLD1, shRNA-scramble, shRNA-N-Ras, and shRNA-KRas constructs were described and validated previously (Kafri et al., 2005; Mor et al., 2007b, 2009).
Cell culture, transfection, and stimulation Jurkat T cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 1% penicillin/streptomycin (Biological Industries, Kibutz Beit Haemek, Israel). Panc-1 cells (American Type Culture Collection) were grown in DMEM containing 10% fetal calf serum, 2 mM L-glutamine, and 1% penicillin/streptomycin. The cells were incubated at 37 1C in a humidied atmosphere of 95% air and 5% CO2. Transfection of Jurkat cells was performed with DMRIE-C (Invitrogen Corporation/ Molecular Probes), and cells were examined at 24 to 48 hours later. Jurkat T cells were serum starved at 37 1C for 2–6 hours, followed by stimulation with 5 mg ml1 of mouse anti-human CD3.
Glutathione S-transferase pull-down assay Detection of activated Ras and Rap1 was performed as described previously (Mor et al., 2007b, 2009).
SDS-PAGE and immunoblotting Samples were separated by SDS-PAGE using 10% polyacrylamide gels and transferred to nitrocellulose filters. Blots were blocked for 1 hours in TBST (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20) containing 3% serum bovine albumin, followed by overnight incubation at 4 1C with the primary antibodies. Blots were washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using the LAS-3000 imaging system (Fujifilm, Tokyo, Japan).
Adhesion assay Jurkat T-cell adhesion to ICAM-1-coated plates was performed as described previously (Mor et al., 2009). Recombinant ICAM-1 was produced as described previously (Mor et al., 2007b). Cells were labeled with 5-carboxyuorescein. Cells (1.5 105) were plated for 20 minutes before removal of non-adherent cells by serial washes. The percentage of adherent cells was quantified with a plate reader (Synergy HT, BioTek Instruments, Winooski, VT) reading emissions at 525 nm.
Microscopy Live cells were plated in 35-mm dishes containing a number 0 glass coverslip over a 15-mm cutout (MatTek, Ashland, MA). Cells were maintained at 37 1C using a PDMI-2 microincubator (Harvard Apparatus, Holliston, MA). Individual cells were imaged before and after addition of stimuli. Images were acquired with a Zeiss 510 inverted laser scanning confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY) and processed with Adobe Photoshop CS.
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Mice The Institutional Ethics Committee of Tel Aviv University approved the study. BALB/c female mice at 8 to 10 weeks of age were used. Oral administration of FTS-A required doses of 50 and 100 mg kg1 (Barkan et al., 2006). DNFB and olive oil were obtained from SigmaAldrich. Mice were sensitized on the shaved abdomen with 50 ml of 0.5% DNFB in a vehicle of 4:l acetone:olive oil. Mice were ear challenged with 20 ml of 0.2% DNFB in a vehicle of 4:l acetone:olive oil after 5 days. A constant area of the ears was measured immediately before challenge and at 24 hours later with an engineer’s micrometer (Ozaki Mfg, Itabashi, Tokyo, Japan). Ear swelling was expressed as the difference in ear thickness before and after the challenge (Zimber et al., 1982). Betamethasone 0.05% (W/W) was used as a positive control (NYU School of Medicine, NY).
Histology and immunohistochemistry Mice ears were fixed in 4% paraformaldehyde, embedded in paraffin, and cut in 4-mm-thick sections. Sections were stained with hematoxylin and eosin, and with specific primary antibodies as mentioned above. Bounding antibodies were detected with peroxidase-based staining. As a negative control, sections were stained in the absence of primary antibody as well as with isotype control sera. Toluidine blue staining protocol was adopted from NYU core facility (http://cores.med.nyu.edu).
Cytokines levels Levels of serum TNF-a were determined at 48 hours after epicutaneous challenge using ELISA kit, according to the manufacturer’s instructions (TNF-a HS ELISA kit, R&D Systems, Minneapolis, MN).
Statistical analysis Significant differences between the control and variable conditions in each experiment were determined by unpaired or paired Student’s t-test, as appropriate using Microsoft Excel. A P-value of p0.05 was considered significant. Data are given as mean±s.e.m. CONFLICT OF INTEREST The authors state no conflict of interest.
ACKNOWLEDGMENTS This research was supported by the Morasha Program of the Israel Science Foundation (grant no. 1822/07). Adam Mor has received grant support from the Israel Science Foundation. This work was supported in part by a grant from the Israel Science Foundation to Yoseph Mekori, who is the incumbent of the Reiss Chair in Dermatology, Tel Aviv University. Yoel Kloog is the incumbent of the Skirball Chair in Applied Neurobiology, Tel Aviv University, and is supported by the Prajs-Drimmer Institute for Anti-degenerative Disease Drugs. We would like to thank Luis Chiriboga, PhD, for slide preparation. We would like to acknowledge Dr Mark Philips (NYU) for excellent and substantial scientific and technical support.
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