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* Division of Pulmonary, Allergy and Critical Care Medicine and
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and
Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06511
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heme oxygenases (HO)3are the rate-limiting enzymes in heme degradation, catalyzing the cleavage of the heme ring to form ferrous iron, carbon monoxide (CO), and biliverdin (1). A catalytic by-product of HO enzyme activity that has received increasing attention in recent years is CO. CO is a gaseous molecule with known toxicity and lethality to living organisms exposed to industrial doses. However, despite this known paradigm of CO toxicity, there has been renewed interest in recent years in the role of CO as a signaling and regulatory molecule in cellular and biological processes (2, 3, 4, 5, 6, 7). Mammalian cells have the ability to generate endogenous CO primarily through the catabolism of heme by HO, with a much smaller amount produced by the peroxidation of lipids (8). Exogenous CO has been shown to have potent anti-inflammatory and antiapoptotic effects (1, 8, 9, 10, 11, 12). Recently, our laboratory has also demonstrated that CO confers potent antiproliferative effects in airway smooth muscle cells (13) and in vascular smooth muscle cells (14). These antiproliferative effects of CO in smooth muscle cells were dependent on mitogen-activated protein kinase (MAPK) and cGMP signaling pathways (13, 14).
Proteases of the caspase family constitute the central executioners of apoptosis. Several recent observations suggest that caspases and apoptosis-regulatory molecules exert important functions beyond that of cell death, including the control of T lymphocyte proliferation and cell cycle progression (15). Data have emerged to suggest that caspases play an important role not only as initiator and effector molecules in the apoptotic signaling cascade, but also in T lymphocyte activation and proliferation (16, 17, 18, 19, 20). Miossec et al. (16) were the first to report that caspase-3-like activity was present in nonapoptotic proliferating T lymphocytes stimulated with PHA. Others have since documented increased caspase activity in proliferative responses to superantigens and alloantigens in vitro and to staphylococcal enterotoxin B in vivo (17). Indeed, in primary T lymphocyte cultures both peptide-based caspase inhibitors and overexpressed endogenous caspase inhibitors significantly blunted proliferative responses to a variety of stimuli (19, 20).
The observation that HO-1/CO inhibits caspase activity in several cell types (21, 22) raised the possibility that CO can regulate lymphocyte proliferation by inhibition of caspases. We conducted experiments to test the hypothesis that CO-induced inhibition of T lymphocyte proliferation is dependent on caspase activity, particularly caspase-3 and caspase-8, and independent of the known CO-regulated pathways such as cGMP and MAPK.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6 and p21-/- and p21+/+ wild-type control mice were obtained at 6–8 wk of age from The Jackson Laboratory (Bar Harbor, ME). Mkk3-/-, Mkk3+/+, Jnk1-/-, and Jnk1+/+ mice were bred at the University of Pittsburgh. Use of animals was approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
Cell preparation and culture
Primary T lymphocytes were isolated from spleens of mice with an Ab-coated column (R&D Systems, Minneapolis, MN) to yield a >95% CD3+ population assessed by flow cytometry using FACScan (BD Biosciences, Mountain View, CA). Cells were cultured in 96-well plates at 50 x 103 cells/well in triplicate in RPMI 1640 medium supplemented with 10% FBS, 10 mM HEPES, 13.6 µM folic acid, 0.3 mM L-asparagine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM L-glutamine. Jurkat (clone E6-1) was purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT). A caspase-8-deficient Jurkat cell line JB6 (23) was originally obtained from Dr. S. Nagata (Osaka University, Osaka, Japan) and maintained in RPMI 1640 as described previously (24).
Reagents
The guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1 (ODQ, 10–100 mM; Calbiochem-Novabiochem, San Diego, CA) was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO). Caspase family inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, and caspase-8 inhibitor Z-IETD-FMK were purchased from Biovision (San Diego, CA).
CO exposure
For cell culture experiments, CO at a concentration of 1% (10,000 parts per million (ppm)) in compressed air was mixed with compressed air containing 5% CO2 in a stainless steel mixing cylinder before being delivered into the exposure chamber. Flow into the chamber was at a rate of 2 L/min. The chamber was humidified and maintained at 37°C. A CO analyzer (Interscan, Chatsworth, CA) was used to measure CO levels in the chamber and there were no fluctuations in the CO concentrations after the chamber had equilibrated.
Cell counts and [3H]thymidine incorporation
Cells were stimulated to proliferate with immobilized anti-CD3 Ab (25 µg/ml; BD Biosciences). Cells were counted daily using a Neubauer hemocytometer (VWR Scientifics, West Chester, PA). Viability was assessed with trypan blue exclusion methods. Proliferation was assessed by tritiated thymidine ([3H]TdR) (New England Nuclear, Boston, MA) incorporation for the final 6 h before culture harvest and measured by scintillation spectroscopy. Data are presented as the mean counts per minute per well. Experiments were done in quadruplicate. In caspase inhibitor experiments, cells were treated at the time of plating with indicated concentrations of caspase inhibitor or an equivalent dilution of the stock solvent DMSO.
Annexin V/propidium iodide (PI) staining
Using the Annexin VFITC kit from BD PharMingen (San Diego, CA), we followed the manufacturer’s protocol. Briefly, T lymphocytes were washed with cold PBS and resuspended with binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2) before transferring 1 x 105 cells to a 5-ml tube. Then 5 µl of annexin V and 5 µl of PI were added, and cells were incubated for 15 min in the dark. Binding buffer (400 µl) was then added to each tube and analyzed by flow cytometry.
cGMP immunoassays
Cellular levels of cGMP were quantified using a commercially available immunoassay (Biomol, Plymouth Meeting, PA). T lymphocytes were incubated in the presence or absence of CO (250 ppm) or ODQ, a selective inhibitor of soluble guanylate cyclase, and cell lysates were analyzed for cGMP content as suggested by the vendor.
Caspase/5-bromo-2-deoxyuridine (BrdU) costaining
To assess caspase activity in proliferating cells, cultures were stained with cell-permeable fluorogenic active caspase indicator, FAM-Val-Ala-Asp-fluoromethylketone (Intergen, Purchase, NY) and an analog of the DNA precursor, BrdU (BD Biosciences) according to kit instructions and analyzed by flow cytometry.
Caspase activity assay
Protein lysates were obtained from T lymphocytes cultured in six-well plates at 2.5 x 106 cells/ml in a final volume of 6 ml and stimulated with immobilized anti-CD3 Ab (25 µg/ml). For the caspase activity assay, cells were washed in PBS and lysed by freeze-thaw in buffer A (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM EGTA). Cell lysates were obtained from supernatants after a 30-min centrifugation at 13,000 rpm at 4°C. Protein concentration was measured using the BCA Protein Assay kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. Fifteen micrograms of protein samples were added to 100 µM caspase-3 substrate, acetyl-DEVD-4-p-nitroanilide (Alexis, San Diego, CA) and OD was assessed over time by colorimetry at 400 nm using a spectrophotometer (Molecular Devices Spectromax 40 Microplate Reader; Molecular Devices, Sunnyvale, CA). Caspase-8 activity was measured using a Caspase-8 Colorimetric Activity Assay kit (Chemicon International, Temecula, CA). Activity (U) is presented as the change in OD (
OD) per milligram of protein per hour over the linear portion of the OD vs time line.
Cell extracts and Western Blot analysis
Cells were harvested and washed in ice-cold PBS and immediately resuspended in cell lysis buffer with complete protease inhibitor mixture (New England Biolabs, Beverly, MA). Cellular protein extracts were electropheresed under denaturing conditions (10–12.5% polyacrylamide gels) and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). Caspase-3 and p2lCipl was detected using a rabbit anti-human polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA). 
-Actin was detected using mouse anti-human -actin mAb (Sigma-Aldrich). Primary Abs were detected using HRP-conjugated donkey anti-rabbit or anti-mouse IgG secondary Abs (Pierce). Peroxidase was visualized using the ECL assay (Amersham Life Science., Arlington Heights, IL) according to the manufacturer’s instructions. When indicated, membranes were stripped (62.5 mM Tris · HCl (pH 6.8), 2% SDS, and 100 mM 2-ME, 30 min, 50°C).
Statistical analysis
Data are expressed as the mean ± SE. Differences in measured variables between experimental and control group were assessed using Student’s t test. Statistical difference was accepted at p < 0.05.
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Results |
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We first established the model of T lymphocyte proliferation in vitro using the well-described anti-CD3 Ab stimulation of lymphocyte proliferation. Primary T lymphocytes isolated from mouse spleen were stimulated with anti-CD3 Ab and, as expected, T lymphocyte proliferation increased at 24 and 48 h after stimulation as assessed by the thymidine incorporation assay (Fig. 1).
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It is well established that CO mediates a variety of cell functions including cell proliferation via the guanylyl/cGMP pathway (8, 14, 25, 26). To investigate whether CO mediates its antiproliferative effects in T lymphocytes via this pathway, we first confirmed that the cGMP level was increased in T lymphocytes at 16 h after CO treatment (Fig. 3A) and ODQ, a selective inhibitor of soluble guanylate cyclase, was effective in blocking the increase of cGMP (Fig. 3A). We then treated the cells with ODQ 1 h before exposing the cells to 250 ppm CO. The ability of CO to suppress T lymphocyte proliferation was not reversed by ODQ (Fig. 3B), indicating that the antiproliferative effect of CO is independent of the activation of guanylate cyclase or the generation of cGMP.
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We also investigated whether the MAPK pathways were involved in the antiproliferative effect of CO, as was previously shown in our laboratory (13, 14). We harvested T lymphocytes from mice in which Mkk3, an upstream activator of p38, and Jnk1 gene was deleted. T lymphocytes from the wild-type strain matched mice were used as controls. In the presence of 250 ppm of CO, the CD3-stimulated cell proliferation was markedly inhibited in cells isolated from gene-deleted mice for Mkk3 and Jnk1 observed in control cells isolated from negative littermates for Mkk3 and Jnk1 (Fig. 3C), indicating that the antiproliferative effect of CO is independent of the Mkk3 and Jnk1 MAPK signaling pathways.
Caspase inhibition prevents lymphocyte proliferation
To determine the role of caspase activity in T lymphocyte proliferation, we applied caspase/BrdU double staining to evaluate the caspase activity in proliferating cells. We demonstrated a correlation between BrdU-positive proliferating cells and cells staining for pan-caspase (Fig. 4A), indicating that caspase activity is strongly related to proliferation.
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CO inhibited caspase activity
Because of the important role of caspases in T lymphocyte proliferation, we studied the effect of CO on caspases. We demonstrated that CO inhibited caspase-3 activity significantly at 24 h (83.4%) and 48 (33.7%) h after CD3 stimulation of primary T lymphocytes (Fig. 5A). The inhibition of caspase-3 by CO was confirmed by Western blot analysis (Fig. 5B). We also evaluated the effect of CO on caspase-8. Caspase-8 activity assay demonstrated that CO inhibited caspase-8 by 49.1% 48 h after CD3 stimulation (Fig. 6).
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To assess the role of CO on cell cycle protein expression, we then examined the protein level of p21Cip1, a potent inhibitor against cell cycle progression, by Western blot analysis. Forty-eight hours after CD3 stimulation, cells exposed to CO demonstrated an increase in p21Cip1 (Fig. 7A). To further investigate the role of caspase in proliferation, we compared the decrease of pan-caspase activity by CO in the T lymphocytes cultured from p21Cip1 knockout and wild-type mice. In T lymphocytes obtained from wild-type mice, CO inhibited caspase activity by 40%. In T lymphocytes from p2Cip1 knockout mice, however, CO inhibited caspase activity by <20% (Fig. 7B). Taken together, these data suggest that CO-mediated inhibition of caspase activity is partially mediated by up-regulation of p21Cip1.
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Having demonstrated that CO inhibited both caspase-3 and -8 activity and the important role of caspases in proliferation, we exposed JB6, a Jurkat cell line in which caspase 8 was genetically deleted, to 250 ppm of CO. Proliferation of JB6 cell cultures 48 h after stimulation with serum was decreased by 75% in comparison to wild-type Jurkat control cells in room air. Interestingly though, while the exposure of wild-type cultures to CO decrease proliferation by > 50%, proliferation of JB6 cultures was unaffected by exposure to CO (Fig. 8A). These data suggest that the antiproliferative effect of CO is dependent on caspase-8. To confirm this finding, we exposed primary murine T lymphocytes stimulated to proliferate with anti-CD3 Ab to the caspase-8-specific inhibitor Z-IETD-FMK. In cultures in which caspase-8 was pharmacologically blocked, CO had no effect on T lymphocyte proliferation (Fig. 8B). These data provide genetic and pharmacological support indicating that the antiproliferative effect of CO is dependent on caspase-8.
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Discussion |
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Exposure to a high concentration of CO can be lethal in the context of industrial or accidental exposure (29). Against this paradigm of CO toxicity, we and others have recently reported that a low concentration of CO can act as a signaling molecule in cellular and biological processes and can confer cytoprotection against a variety of cellular stresses (1, 12, 13, 14, 28, 30). The concentration of CO used for our studies is less than one-tenth of the CO concentration used in humans during measurement of DLCO in pulmonary function testing (e.g., 3000 ppm CO used in DLCO tests). Previous studies also demonstrated that long-term (2 years) exposure of rodents to low levels of CO (500 ppm) produced no significant alterations in physiological or biochemical parameters (27, 31). Also, we have not observed any evidence of cellular or tissue toxicity in our previously reported in vitro and in vivo studies (1, 12, 13, 28, 30) using the same level of CO concentration used for this current study.
Our laboratory has demonstrated the critical role of the Mkk3/p38 MAPK pathway in mediating the anti-inflammatory and antiapoptotic effects of CO (9, 10, 12, 30, 32). For example, in endothelial cells the antiapoptotic effect of CO is strictly dependent on the activation of p38 MAPK, independent of the guanylate cyclase/cGMP system (32). However, in fibroblasts the same antiapoptotic effect is dependent on the activation of guanylate cyclase (11). Our laboratory has also examined the effect of CO on vascular smooth muscle cells and found it to have similar effects as HO-1, as growth arrest was induced and mediated via a cGMP and p38 MAPK pathway (14). We also demonstrated that the antiproliferative effect of CO in airway smooth muscle cells is mediated by the extracellular signal-regulated kinase MAPK pathway (13). Most of the biological effects attributed to CO including anti-inflammatory, antiapoptotic, and now antiproliferative effects have been linked to its ability to modulate the activity of guanylate cyclase and to increase the levels of cellular cGMP (7, 14, 25, 26, 28, 33, 34) and/or the MAPK pathways such as p38 or extracellular signal-regulated kinase.
In our present study, however, we were surprised to observe that the antiproliferative effects of CO in T lymphocytes did not involve the aforementioned critical cGMP (6, 7, 25, 26, 33) or MAPK (13, 14, 28) pathways by which CO signals its biological effects, such as the antiproliferative effects in other cell types, including airway and vascular smooth muscle cells. We treated the cells with ODQ, a selective inhibitor of soluble guanylate cyclase 1 h before exposing the cells to 250 ppm CO. However, the ability of CO to suppress T lymphocyte proliferation was not reversed by ODQ (Fig. 3B), indicating that the antiproliferative effect of CO is independent of the activation of guanylate cyclase or the generation of cGMP.
We also observed that the MAPK pathways were not involved in the antiproliferative effect of CO. We harvested the spleen T lymphocytes from Mkk3, which functions upstream of p38, and Jnk1 gene-deleted (-/-) and littermate control mice. In the presence of 250 ppm of CO, the CD3-stimulated cell proliferation was markedly inhibited in both Mkk -/- and Jnk1-/- as well as in control cells (Fig. 3C), indicating that the antiproliferative effects of CO is independent of the p38/Mkk3 and Jnk1 MAPK pathways. Thus, our current study demonstrates the critical importance of cell specificity in CO signaling for its antiproliferative effects. Whether this cell specificity of CO signaling is also observed in its other biological effects, such as its anti-inflammatory or antiapoptotic effects, remains to be seen; however, this current study strongly suggests that cell specificity has to be considered for other biological effects.
Caspase activity was shown to be up-regulated during T lymphocyte proliferation. Low-level caspase-8 and caspase-3 activation was detected in proliferating IL-7-treated cells in the absence of cell death during the first days of culture (34). Caspase inhibitors suppressed IL-7-induced expansion of recent thymic emigrants (34). The caspase-8 inhibitor c-FLIPL modulates TCR-induced proliferation but not activation-induced cell death of lymphocytes (35). Others also reported that CD3-induced proliferation and IL-2 production by human T cells are blocked by inhibitors of caspase activity (18). These findings extend the role of death receptors to the promotion of T lymphocyte growth in a caspase-dependent manner.
Since our data strongly suggest that CO confers its antiproliferative effects in T lymphocytes independently of cGMP and the MAPK pathways, we then sought to determine whether there was a mechanistic link between CO-mediated inhibition of caspase activity and the antiproliferative activity of CO in lymphocytes. Our data demonstrated that caspase activity was up-regulated during the process of T lymphocyte proliferation and that this proliferation was blocked with the pan-caspase inhibitor, Z-VAD (Fig. 4B). These data are consistent with the observation of other laboratories that implicate caspases in lymphocyte proliferation (16, 17, 18, 19, 20). Stimulation of T lymphocytes to proliferate results in a heterogeneous population of cells that are alive and proliferating as well as dying, whether by neglect (from lack of TCR stimulation) or Ag-induced cell death (from repeated TCR stimulation). Therefore, it is possible that the increase in caspase activity seen after T lymphocyte stimulation came from the apoptotic fraction of stimulated cultures. To address this, we performed caspase/BrdU double staining to evaluate the caspase activity in proliferating cells. We demonstrated that proliferating cells (BrdU positive) are also caspase positive (Fig. 4A), indicating that caspase activity is strongly related to proliferation.
A total of 14 caspase family members has been identified in humans. In this report, we focused on caspase-3- and -8-like activity and cleavage because caspase-3 is a prototypic downstream effector protease and caspase-8 is the most upstream initiator caspase (36). We demonstrated that CO inhibited caspase-3 activity significantly at 24 h (83.4%) and 48 (33.7%) h after CD3 stimulation of primary T lymphocytes (Fig. 5A). The inhibition of caspase-3 by CO was confirmed by Western blot analysis (Fig. 5B). We also evaluated the effect of CO on caspase-8. Caspase-8 activity assay demonstrated that CO inhibited caspase 8 by 49.1% 48 h after CD3 stimulation (Fig. 6).
Having demonstrated that CO inhibited both caspase-3 and -8 activity and recognizing the important role of caspases in proliferation, we exposed JB6, a Jurkat cell line in which caspase-8 was genetically deleted, to 250 ppm of CO. Proliferation of JB6 cell cultures 48 h after stimulation with serum was decreased by 75% in comparison to wild-type Jurkat controls in room air. Interestingly, although exposure of wild-type cultures to CO decreased proliferation by >50%, proliferation of JB6 cultures was unaffected by exposure to CO (Fig. 8A). These data suggest that the antiproliferative effect of CO is dependent on caspase-8. To confirm this finding, we exposed primary murine T lymphocytes stimulated to proliferate with anti-CD3 Ab to the caspase-8-specific inhibitor Z-IETD-FMK. In cultures in which caspase-8 was pharmacologically blocked, CO had no effect on T lymphocyte proliferation (Fig. 8B). These data provide genetic and pharmacological support indicating that the antiproliferative effect of CO is dependent on caspase-8.
Cyclin-dependent kinase inhibitors such as p21Cip1, p16Ink4, and p27Kip1 are implicated in the regulation of cyclin-cyclin-dependent kinase activity (37). p21Cip1 is up-regulated in arteries after vascular injury and the overexpression of p21Cip1 in vascular smooth muscle cells results in G1 arrest and inhibition of cell growth (38). In this study, we observed that exposure of T lymphocytes to CO also induced up-regulation of p21Cip1 protein expression strongly by 48 h (Fig. 7A), suggesting that the antiproliferative effect of CO is dependent on the expression of the cell cycle inhibitor p21Cip1. To further investigate whether CO-induced p21Cip1 correlated with the activity of caspase in cell proliferation, we compared the decrease of pan-caspase activity by CO in the T lymphocytes cultured from p21Cip1 knockout to wild-type mice. We demonstrated that in the T lymphocyte from p21Cip1 knockout mice, the decrease of pan-caspase activity was significantly inhibited (Fig. 7B), indicating that the antiproliferative effect of CO is partially associated with increased expression of the cell cycle inhibitor p21Cip1.
In summary, our data suggest that CO could inhibit CD3-activated T lymphocyte proliferation. This was associated with an inhibition of caspase activity and activation, independent of cGMP and MAPK pathways. Interestingly, we observe that CO’s ability to confer cell growth arrest is highly dependent on caspases, especially caspase-8 and caspase-3, which are regulated by p21Cip1. Subsequent delineation of the identity and function of the caspase or caspase-like molecule(s) involved in lymphocyte proliferation will provide insight into regulation of signaling molecules that are involved with both cell death and proliferation. The elucidation of the signaling pathways involved in the antiproliferative effects of CO is important not only for our basic understanding of the mechanism of action of CO, but also in the search for novel targets for new therapeutic approaches to regulate T lymphocyte function in vivo in pathophysiological states.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Augustine M. K. Choi, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, MUH628, 3459 5th Avenue, Pittsburgh, PA 15213. E-mail address: choiam{at}msx.upmc.edu
3 Abbreviations used in this paper: HO, heme oxygenase; HO-1, heme oxygenase-1; CO, carbon monoxide; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; Jnk, c-Jun NH2-terminal kinases; ppm, parts per million; ODQ, 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1; BrdU, 5-bromo-2-deoxyuridine; PI, propidium iodide, Z-VAD, benzyloxycarbonyl-Val-Ala-Asp; Z-IETD-FMK, benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone.
Received for publication June 27, 2003. Accepted for publication November 5, 2003.
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References |
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mitogen-activated protein kinase pathway during ischemia-reperfusion lung injury. J. Biol. Chem. 278:22061.-induced apoptosis in cultured fibroblasts. Am. J. Physiol. 278:L312.
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, CO.
, Room air; 
, 250 ppm CO. C, Primary spleen T lymphocytes were harvested from Mkk3+/+, Mkk3-/-, Jnk1+/+, and Jnk1-/- mice. Proliferation was stimulated with immobilized anti-CD3 in the presence and absence of 250 ppm CO and assessed 48 h after stimulation by [3H]TdR incorporation. Results are depicted as mean ± SE cpm of conditions in triplicate (*, p < 0.05 vs room air control). 
, Z-VAD.
