Introduction

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Zinc tetrakis-(N-methyl-4'-pyridyl) porphyrinato (ZnTMPyP) is a porphyrin analog having an extensive conjugated ring system that undergoes reversible one-electron oxidation. Zinc porphyrins have been reported to exhibit anti-inflammatory and anti-allergic properties in both in vitro and in vivo models as an interleukin (IL)-1 antagonist or as an inhibitor of heme-dependent enzymes, such as guanylate cyclase and nitric-oxide synthase (Luo and Vincent, 1994; Okada, 1996; Zhao et al., 1996). In our previous study, ZnTMPyP has been reported to be a potent inhibitor in reactive oxygen species (ROS) production by RAW 264.7 macrophages in response to various stimulants, and also was effective in reducing oxidative stress-induced DNA-binding activity of NF-B and IL-1 production (Kang et al., 2001). Zinc protoporphyrin has also been shown to inhibit mitogen-induced lymphocyte proliferation (Nagai et al., 1992).

The proliferation of T cells involves a complex process with multiple signal transduction pathways (Krummel and Allison, 1995). Activation of T cell receptor (TCR)-CD3 complex and/or IL-1 receptor (IL-1R) generates intracellular signals that transiently increase the transcription of several genes, including growth-promoting cytokines. Among them, the induction of IL-2 is the hallmark of T cell proliferation, and the expression of the IL-2 gene is regulated at the transcription level with subsequent increased secretion of IL-2, which acts as an autocrine/paracrine factor important for the sustained proliferation of activated T cells (Roy et al., 1997).

Several observations have suggested a role for NF-B in cell proliferation through regulation of growth-promoting cytokines, such as IL-2, IL-4, and IL-6 (Ghosh et al., 1993; Mora et al., 2001) or cell cycle-dependent kinases (Hinz et al., 1999). The predominantly characterized NF-B complex is a p50-p65 heterodimer, which at rest is retained in the cytoplasm and is associated with an inhibitor molecule, IB (Zabel and Baeuerle, 1990). In response to a variety of stimuli, IB is phosphorylated and becomes dissociated from the NF-B complex. Free NF-B can then translocate to the nucleus where it binds to the NF-B motif and functions as a transcriptional regulator. Although the molecular mechanisms of NF-B activation in response to mitogenic stimulation remain to be elucidated, there is recent evidence identifying ROS or mitogen-activated protein kinases (MAPKs) as important modulators of the signaling cascade initiated by mitogens in T cells and thymocytes (Schafer et al., 1999; Tatla et al., 1999; Zhang et al., 1999; Pani et al., 2000). However, the specific mechanisms responsible for ZnTMPyP-induced changes of T cell activation have not been illustrated.

The objective of the present study was to determine whether ZnTMPyP exhibited the ability to inhibit thymocyte proliferation, IL-2 production, DNA binding activity of NF-B, the activation of MAPK, ROS production, and tyrosine phosphorylation after mitogenic stimulation of thymocytes.

	Materials and Methods

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Reagents. ZnTMPyP was purchased from Mid-Century (Posen, IL). Concanavalin A (Con A), lipopolysaccharide (LPS) from Escherichia coli serotype 055B5, SB203580, and PD98059 were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant mouse IL-1 was purchased from Serotec Ltd. (Oxford, UK). DNA polymerase and deoxynucleoside-5'-triphosphate were purchased from Invitrogen (Carlsbad, CA). Antibodies used in this study were anti-rabbit phospho-p38 MAPK/MAPK antibody (New England Biolabs, Beverly, MA) and anti-phosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY).

In Vitro ZnTMPyP Treatment of Thymocytes. Thymus glands from male CD-1 mice (4-5 weeks of age) were aseptically removed, and cell suspensions were prepared in RPMI 1640 with 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 100 units/ml mycostatin, 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT), and 2 × 10^-5 M mercaptoethanol. Cells were counted using an electronic cell counter (Beckman Coulter Inc., Fullerton, CA). Thymocytes were treated with various concentrations of ZnTMPyP (0-100 µM) for 2 h before the addition of a mitogenic factor. The cultures were then incubated for various periods of time as indicated.

Cell Line and Cell Culture. To obtain LPS-exposed macrophage supernatant having IL-1-like mitogenic activity, RAW 264.7 cells, a mouse peritoneal macrophage cell line (American Type Culture Collection, Manassas, VA), were resuspended in RPMI 1640 (Mediatech, Herndon, VA) containing 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 100 units/ml mycostatin, and 10% fetal bovine serum. Aliquots of 1 ml, containing 10⁶ cells, were added to 24-well plates (Costar, Cambridge, MA) and incubated at 37°C in a humidified atmosphere of 5% CO₂ for 2 h. The nonadherent cells were then removed by vigorously washing twice with 1 ml of RPMI media. The adherent cells were further incubated in 1 ml of RPMI media containing 5 µg/ml LPS. After incubating the cell culture for 24 h, the supernatant was collected, filtered, and stored at 70°C until the thymocyte proliferation assay was performed.

Measurement of Cell Viability. Lactate dehydrogenase (LDH) is an abundant intracellular enzyme, and its release into cell culture supernatants is a marker of lytic cell death (Lipton et al., 1993). The activity of LDH was measured using a LDH determination kit (Roche Diagnostics, Mannheim, Germany). Briefly, 100 µl of ZnTMPyP (1-100 µM) were added to 100 µl of thymocytes (10⁶/ml) in given wells of a microplate. The cells were then incubated at 37°C in a humidified atmosphere of 5% CO₂ for 1 to 72 h. After incubation, 100 µl of supernatant were added to 100 µl of reaction mixture and incubated for 30 min at room temperature. Absorbency of the samples at 490 nm was measured using a microplate reader. Results were expressed as percentage of cell viability, referenced to the maximum LDH released when cells were lysed with detergent, using the formula, % viability = 100 × (1  [experimental  untreated)/(detergent  untreated)]).

Thymocyte Proliferation Assay. Direct inhibitory effects of ZnTMPyP on thymocyte proliferation in response to various mitogenic factors were studied according to the method of Kang et al. (1992). Briefly, an aliquot of 100 µl of Con A (2.5 µg/ml), IL-1 (10 ng/ml), or LPS-exposed macrophage supernatant was added in quadruplicate to 96-well microculture plates and 100 µl of thymocyte suspension in the presence of ZnTMPyP (0-100 µM). After a 54-h incubation at 37°C in 5% CO₂, the cultures were pulsed for 18 h with [³H]thymidine (1.0 µCi/well; activity: 2.0 Ci/mmol; PerkinElmer Life Sciences, Boston, MA) and harvested using a cell harvester (Brandel Inc., Gaithersburg, MD). The radioactivity in the collecting glass filter disks was measured using a liquid scintillation counter (Beckman Coulter).

Nuclear Extracts. Thymocytes (5 × 10⁶) were pretreated with various inhibitors, such as ZnTMPyP (0-100 µM), SB203580 (0-30 µM), and PD98059 (0-50 µM) in 5 ml of RPMI media. After a 1- or 2-h pretreatment, cells were cultured with Con A (2.5 µg/ml) or IL-1 (10 ng/ml) in the absence or presence of each of inhibitor for l h. At the end of the exposure, the cells were harvested and resuspended in hypotonic buffer A [100 mM HEPES (pH 7.9), 10 mM KCl, 0.1 M EDTA, 0.5 mM dithiothreitol, 1% Nonidet P-40, and 0.5 mM phenylmethylsulfonyl fluoride ] for 10 min on ice and then vortexed for 10 sec. Nuclei were pelleted by centrifugation at 12,000g for 30 s and were resuspended in buffer C [20 mM HEPES (pH 7.9), 20% glycerol, 0.42 M NaCl, 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride] for 30 min on ice. The supernatant containing nuclear proteins was collected by centrifugation at 10,000g for 2 min and stored at 70°C.

Electrophoretic Mobility Shift Assay (EMSA). Binding reaction mixtures (10 µl), containing 5 µg (4 µl) of nuclear extract protein, 2 µg of poly(dI-dC) · poly(dI-dC) (Sigma), and 40,000 cpm of ³²P-labeled probe in binding buffer [4 mM HEPES (pH 7.9), 1 mM MgCl_2, 0.5 mM DTT, 2% glycerol, and 20 mM NaCl], were incubated for 30 min at room temperature. Protein-DNA complexes were separated on 5% nondenaturing polyacrylamide gels in 1× Tris-borate/EDTA electrophoresis buffer and autoradiographed overnight. Autoradiographic signals for activated NF-B were quantitated by densitometric scanning using an UltroScan XL laser densitometer (model 2222-020, LKB, Bromma, Sweden) to determine the intensity of each band.

ELISA for IL-2. Thymocytes (10⁶ cells) were incubated in 1 ml of RPMI media containing Con A (2.5 µg/ml) or IL-1 (10 ng/ml) with or without ZnTMPyP in the concentration range of 0-100 µM at 37°C in a humidified atmosphere of 5% CO₂ for 24 h. After 24 h, the supernatant was collected and quantified for IL-2 using a mouse ELISA system (BioSource International, Camarillo, CA).

Western Blotting. Immunoblotting for tyrosine phosphorylation of proteins or phosphorylation of p38 kinase was carried out as described in the protocol supplied by the manufacturer, using phospho-specific antibodies against phosphorylated sites of tyrosine or p38 MAPK. Non-phospho-specific antibodies against p38 MAPK provided in the assay kit were used to normalize the phosphorylation assay using the same transferred membrane blot.

Measurement of Chemiluminescence Generation. ROS production by thymocytes in response to Con A was determined by measuring cellular chemiluminescence using a Berthold luminometer (model LB9505AT; EG&G Berthold, Wildbad, Germany). Briefly, cells were washed once with phosphate-buffered saline (145 mM NaCl, 5 mM KCl, 1.9 mM NaH₂PO₄, 9.35 mM Na₂HPO₄, and 5.5 mM glucose, pH 7.4), centrifuged at 500g and 4°C for 5 min, and resuspended in HEPES-buffered medium (145 mM NaCl, 5 mM KCl, 10 mM NaHEPES, 5.5 mM glucose, and 1 mM CaCl₂, pH 7.4). Cell counts were determined using an electronic cell counter equipped with a cell sizing attachment (Beckman Coulter).

Statistics. Values were expressed as means ± standard errors. Data were analyzed using one-way analysis of variance and Student's t test. Significance was set at p < 0.05.

	Results

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It was essential to demonstrate that ZnTMPyP was not cytotoxic under the condition used for the functional assays employed in this study. Viability of thymocytes was monitored as LDH activity in the acellular culture media at rest. Membrane integrity of thymocytes was not compromised after 1, 3, 24, or 72 h of in vitro exposure to ZnTMPyP (1-100 µM); i.e., ZnTMPyP did not increase the activity of LDH in the culture media (data not shown).

To verify that ZnTMPyP-mediated suppression of the mitogenic response was a direct effect on thymocytes, these cells were pretreated for 2 h with different concentrations of ZnTMPyP and then examined for proliferation activity of cells by treatment with various mitogenic factors, such as Con A, IL-1, or LPS-exposed macrophage supernatant. Con A, IL-1, or LPS-exposed macrophage supernatant enhanced thymocyte proliferation by 86-, 7.5-, and 107-fold, respectively. The data shown in Fig. 1, A to C, indicate that ZnTMPyP directly inhibited the mitogenic factor-stimulated thymocyte proliferation in a concentration-dependent manner with a maximum inhibition of 94 and 77% for Con A or LPS-exposed macrophage supernatant, respectively, at 100 µM ZnTMPyP, and 100% for IL-1 at 50 µM ZnTMPyP. In contrast, ZnTMPyP had no effect on the basal rate of [³H]thymidine incorporation in unstimulated thymocytes (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Direct effect of ZnTMPyP (1-100 µM) on thymocyte proliferation in response to Con A (2.5 µg/ml; A), IL-1 (10 ng/ml; B), or LPS-exposed macrophage supernatant (C). Stimulant-induced thymocyte proliferation was calculated as cpm of [3H]thymidine incorporation of mitogen-treated thymocytes minus that with resting cells. Values are means ± standard errors of three separate experiments. , significant decrease (p < 0.05) compared with stimulant alone.

NF-B has been implicated in the regulation of cell proliferation by controlling the expression of growth-promoting cytokines. Among them, the induction of IL-2 is the hallmark of T cell proliferation (Roy et al., 1997). Therefore, to address the mechanism of ZnTMPyP-mediated modulation of T cell activation/proliferation, the effects of ZnTMPyP on NF-B activation and IL-2 production in mitogen-stimulated thymocytes were examined. Con A or IL-1 increased the DNA binding activity of NF-B by 2- and 1.8-fold, respectively, compared with unstimulated cells. ZnTMPyP completely inhibited DNA binding activity of NF-B induced by Con A or IL-1 at 1 µM ZnTMPyP (Fig. 2, A and B). To confirm that protein binding to the NF-B motif was due to NF-B (p50 and p65 heterodimer), supershift assays were performed. The data indicate that NF-B heterodimers (p50/p65) were activated in thymocytes stimulated with Con A or IL-1, since supershift was detected with antibodies to p50 and p65, with reciprocal decreases in the intensity of the NF-B band. The addition of the cold competitor eliminated the specific bands in the samples from Con A- or IL-1-treated cells, indicating that the band on the autoradiogram was specific for NF-B binding (Fig. 2, C and D).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   EMSA illustrating the effect of ZnTMPyP on Con A-induced (A) or IL-1-induced (B) activation of NF-B. Nuclear extracts were prepared from thymocytes pretreated for 2 h with ZnTMPyP (0-100 µM) and then stimulated with Con A (2.5 µg/ml) or IL-1 (10 ng/ml) for an additional 1 h. The results of EMSA are shown (upper panels) and quantitated by densitometric analysis as a percentage of the response to stimulant alone minus control (lower panels). Supershift EMSA using nuclear proteins of thymocytes are shown 1 h after Con A (C) or IL-1 (D) treatment. The addition of antibodies for p50 and p65 caused supershifts, with a reciprocal decrease in the intensity of the NF-B band. The supershift bands are indicated by arrows. Addition of 100 ng of unlabeled cold competitor to the stimulant samples successfully competed for NF-B binding and eliminated the specific band (lanes 5 of C and D). Data are representative of at least three separate experiments.

ZnTMPyP also inhibited IL-2 production by mitogen-stimulated thymocytes. Con A- or IL-1 enhanced IL-2 production by thymocytes by 10- or 4-fold compared with unstimulated cells, respectively. The enhancement of IL-2 induced by Con A was inhibited by 89% with 50 µM ZnTMPyP, whereas IL-1-stimulated IL-2 production was decreased by 73% with 1 µM ZnTMPyP (Fig. 3, A and B).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Effect of ZnTMPyP on IL-2 production by thymocytes stimulated by Con A (A) or IL-1 (B). Thymocytes (106/ml) were incubated at resting or after stimulation with Con A (2.5 µg/ml) or IL-1 (10 ng/ml) in the presence or absence of ZnTMPyP (50 and 1 µM with Con A- and IL-1-treated cells, respectively). After 24 h, IL-2 production in the supernatant of cells was measured by an ELISA system. Stimulant-induced IL-2 production was calculated as nanograms per milliliter. Values are means ± standard errors of three separate experiments. , significant increase (p < 0.05) compared with control. +, significant decrease (p < 0.05) compared with stimulant alone.

The family of MAPKs, such as p38 MAPK, extracellular signal-regulated kinase (ERK), and Jun NH₂-terminal kinase (JNK), has been demonstrated to play an important role in signaling pathways in activated T cells. Since we found that the activation of p38 MAPK or ERK correlates closely with the mitogenic-stimulated thymocyte proliferation and IL-2 production (data not shown), questions were raised as to whether activation of p38 MAPK or ERK was involved in the signal transduction pathways leading to NF-B activation in response to thymocyte stimulation, and whether ZnTMPyP inhibited this activation of MAPK. To examine the role of p38 MAPK or ERK in the NF-B activation, thymocytes were preincubated with different concentrations of a specific inhibitor of p38 MAP kinase (SB203580, 0-30 µM) or a specific inhibitor of MAP kinase kinase upstream of ERK (PD98059, 0-50 µM) and then examined for NF-B activation induced by Con A (2.5 µg/ml) or IL-1 (10 ng/ml) for 1 h. SB203580 at a higher concentration (30 µM) substantially inhibited the Con A-induced binding activity of NF-B to DNA (45%), whereas IL-1-induced NF-B activation was inhibited over the range of 1 to 30 µM SB203580 by 85 to 100% (Fig. 4, A and B). In contrast, PD98059 failed to inhibit the Con A- or IL-1-induced NF-B activation (Fig. 4, C and D). These results suggest that p38 MAPK, but not ERK, mediates signal integration for NF-B activation-dependent production of IL-2 and thymocyte proliferation in response to mitogenic stimulation, although the p38 MAPK dependence of Con A-induced NF-B activation is partial.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   EMSA illustrating the effect of SB203580 (A and B) or PD98059 (C and D) on Con A- or IL-1-induced activation of NF-B. Nuclear extracts were prepared from thymocytes pretreated for 1 h with SB203580 (0-30 µM) or PD98059 (0-50 µM) and then stimulated with Con A (2.5 µg/ml) or IL-1 (10 ng/ml) for an additional 1 h. The results of EMSA are shown (upper panels) and quantitated by densitometric analysis as a percentage of the response to stimulant alone minus control (lower panels). Data are representative of at least three separate experiments.

Activation of p38 MAPK was observed in cell lysates from Con A-stimulated thymocytes, using Western blot analysis with a phospho-specific p38 MAPK antibody. As with the specific inhibitor of p38 MAPK, ZnTMPyP (50 µM) substantially inhibited the activation of p38 MAPK in response to Con A (2.5 µg/ml) (Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of ZnTMPyP on activation of p38 MAPK in thymocytes in response to Con A. Thymocytes were either unexposed or exposed for 2 h to ZnTMPyP (50 µM) or for 1 h to SB203580 (30 µM) and then stimulated with Con A (2.5 µg/ml) for 10 min. Then Western blots with anti-phospho-p38 MAPK/p38 MAPK antibody were employed to monitor p38 MAPK phosphorylation. Phosphorylation signals and protein abundance signals of p38 MAPK are presented as indicated. Data are representative of at least three separate experiments.

ROS have been reported to be important modulators of the signaling cascade initiated by Con A in thymocytes (Pani et al., 2000). The ability of ZnTMPyP to inhibit the secretion of ROS by thymocytes in response to Con A was determined by measuring the generation of chemiluminescence (Fig. 6). Con A (5 µg/ml) enhanced chemiluminescence generated by thymocytes by 2-fold above the control level. ZnTMPyP (1-50 µM) inhibited Con A-stimulated chemiluminescence in a dose-dependent manner with 100% inhibition at 50 µM.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of ZnTMPyP on chemiluminescence generated by Con A-stimulated thymocytes. The cells (108/ml) were preincubated at 37°C for 10 min and then stimulated with Con A (5 µg/ml) in the presence or absence of ZnTMPyP (0-50 µM). Chemiluminescence was expressed as the integral of cpm versus time minus that generated from resting cells. Values are means ± standard errors of four separate experiments. , significant decrease (p < 0.05) compared with Con A alone.

ROS have been reported to function as physiological regulators of tyrosine phosphorylation via their effects on oxidant-sensitive tyrosine kinase and/or tyrosine phosphatase (Bauskin et al., 1991; Fialkow et al., 1993). Therefore, whether ZnTMPyP, having the ability to inhibit ROS production, also suppressed protein tyrosine phosphorylation was examined in Con A-stimulated thymocytes. An antiphosphotyrosine immunoblot analysis of cell lysates obtained from thymocytes stimulated with Con A (2.5 µg/ml) exhibited marked increase in phosphorylation of multiple proteins, 55, 63, 72, 74, 90, and 106 kDa, after 10 min of stimulation (Fig. 7, lane 2). ZnTMPyP (50 µM) effectively suppressed Con A-induced protein tyrosine phosphorylation (Fig. 7, lane 3).

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Effect of ZnTMPyP on protein tyrosine phosphorylation by Con A-stimulated thymocytes. Thymocytes were either unexposed or exposed for 2 h to ZnTMPyP (50 µM) and then Con A (2.5 µg/ml) for 10 min. Then Western blots with anti-phosphotyrosine antibody were employed to monitor protein tyrosine phosphorylation. Protein sizes are indicated in kilodaltons, and arrows indicate protein bands whose phosphorylation is altered. Data are representative of at least three separate experiments.

	Discussion

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Data presented in this study indicate that ZnTMPyP is an effective in vitro inhibitor of mitogen-stimulated thymocyte activation, i.e., proliferative activity, NF-B activation, IL-2 production, p38 MAPK activation, ROS secretion, and protein tyrosine phosphorylation. These inhibitory actions of ZnTMPyP are not due to cytotoxicity, since ZnTMPyP does not compromise membrane integrity of thymocytes at rest. ZnCl₂ over the range of concentrations from 1 to 100 µM or H₂TMPyP at lower concentrations of 1 to 10 µM exerted little effect on thymocyte proliferation in response to Con A or IL-1, but H₂TMPyP at higher concentrations of 50 to 100 µM caused complete inhibition (data not shown). ZnTMPyP, at concentrations of 1 to 10 µM, exhibited substantial or maximum inhibition of the proliferative activity, suggesting that the binding of Zn²⁺ to the porphyrin moiety strongly enhances the porphyrin-based antiproliferative effect on thymocyte proliferation.

The mechanism by which ZnTMPyP inhibits mitogen-stimulated thymocyte activation has not been resolved. The proliferation of T cells is known to be a complex process involving multiple signal transduction pathways (Krummel and Allison, 1995). Distinct signaling cascades via different cell surface receptors appear to converge on common effector molecules, such as transcription factors and growth-promoting cytokines. NF-B has been shown to play a role in proliferation of T cells via regulation of induction of IL-2 gene expression (Ghosh et al., 1993; Varga et al., 1999). In the present study, mitogenic stimulation of thymocytes induced increases in DNA binding of NF-B as well as subsequent IL-2 production through the activation of multiple receptors, including the CD3 component of the TCR or IL-1R at the T cell surface, respectively. ZnTMPyP substantially inhibited Con A- or IL-1-induced NF-B activation and IL-2 production. These results suggest that ZnTMPyP may block at a common step in the receptor-mediated signal transduction pathway leading to NF-B activation rather than act competitively on mitogen-receptor interaction at the cell plasma membrane. As with thymocytes, inhibition of the activation of NF-B was observed in RAW 264.7 macrophages, and the inhibition was not limited to a single stimulant; i.e., inhibition of silica-, lipopolysaccharide-, and H₂O₂-induced activation was noted (Kang et al., 2001). However, thymocytes seem to be more sensitive to ZnTMPyP than are macrophages; i.e., the complete inhibition of thymocyte NF-B activity was noted at 1 to 10 µM, whereas that for suppression of macrophage activation ranged from 10 to 100 µM.

The molecular mechanism in which ZnTMPyP inhibits NF-B activation is not clear. Several protein kinase cascades have been implied to regulate IL-2 gene transcription for induction of maximal IL-2 synthesis during T cell activation. Among them, we found that the activation of p38 MAPK and ERK correlates closely with the Con A- or IL-1-stimulated thymocyte proliferation and IL-2 production (data not shown). However, the fact that Con A- or IL-1-induced NF-B activation was influenced, respectively, by the specific p38 MAPK inhibitor SB203580 at a high (30 µM) dose versus over a range of concentrations (1-30 µM), but not the specific MAP kinase kinase/ERK inhibitor PD98059 (1-50 µM), suggests that p38 MAPK rather than ERK could be involved in NF-B activation via different receptors. ZnTMPyP also inhibited p38 MAPK activation in response to Con A. In addition to MAPK, ROS, protein tyrosine kinase, and protein tyrosine phosphatase have also been shown to play roles in the activation of NF-B in various cells, including T cells. ROS and protein tyrosine kinase are well known activators of p38 MAPK (Saitoh et al., 1998; Benhar et al., 2001; Haddad and Land, 2002), although the mechanism underlying this activation is not completely known. In our previous study, ZnTMPyP inhibited stimulant-induced ROS production in RAW 264.7 macrophages (Kang et al., 2001). As with macrophages, the data from the present study indicate that ZnTMPyP exhibits the ability to inhibit Con A-induced ROS production as well as protein tyrosine phosphorylation in thymocytes. Overall, the earliest effects of ZnTMPyP seem to be on ROS production and tyrosine phosphorylation. ZnTMPyP could exert inhibitory effects on redox-dependent signaling downstream, such as p38 MAPK, linking NF-B activation in T cells/thymocytes via inhibition of ROS secretion.

ZnTMPyP might have an antioxidant effect due to its ability to undergo reversible one-electron oxidation via its extensive conjugated ring system (Fajer et al., 1970; Dolphin et al., 1971). However, data from our previous study (Kang et al., 2001) indicate that ZnTMPyP (100 µM) exhibits a relatively weak ability to directly scavenge hydroxyl or superoxide radicals, since its potency is much lower than that of well established antioxidants, such as hypotaurine or ascorbic acid, and other metalloporphyrins, such as Mn- or Fe-porphyrins, having the ability by reduction and oxidation to change their valance state. However, 1 or 10 µM ZnTMPyP completely inhibited Con A-stimulated chemiluminescence and NF-B activation. Consistent with our previous data in macrophages, these results suggest that, in addition to an antioxidant effect, ZnTMPyP may directly act as an inhibitor of thymocyte activation.

In conclusion, our findings suggest that the antiproliferative effect of ZnTMPyP is mediated by effective inhibition of ROS production, p38 MAPK activation, NF-B activation, and IL-2 production during mitogenic stimulation of thymocytes. Further studies concerning the effects of ZnTMPyP on the upstream step of NF-B in T cells/thymocytes activated through the TCR complex or IL-1R are warranted.