pathway of CFTR activation also exists in T lymphocytes. Cytosolic ..... autoregulatory role (Kirk et al., 1990; Taylor-Robinson ..... Riordan,J.R. (1993) Annu. Rev.
The EMBO Journal vol.14 no.12 pp.2700-2707, 1995
Activation of CFTR chloride current by nitric oxide in human T lymphocytes
Yan-jie Dong', Anthony C.Chao2, Keisuke Kouyama, Yao-pi Hsu3, Robert C.Bocian3, Richard B.Moss3 and Phyllis Gardner4 Department of Molecular Pharmacology and Medicine and 3Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA 'Present address: Department of Molecular Physiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA 2Present address: ALZA Corporation, 950 Page Mill Road, PO Box 10950, Palo Alto, CA 94303-0802, USA 4Corresponding author A.C.Chao and K.Kouyama contributed equally to this report
Nitric oxide, which is produced by cytokine-activated mononuclear cells, is thought to play an important role in inflammation and immunity. While the function of nitric oxide as a direct cytotoxic effector molecule is well established, its function as a transducer molecule in immune cells is not. By use of whole-cell patch clamp recordings, we show that nitric oxide activates cystic fibrosis transmembrane conductance regulator Cl- currents in normal human cloned T cells by a cGMP-dependent mechanism. This pathway is defective in cystic fibrosis-derived human cloned T cells. These findings not only delineate a novel transduction mechanism for nitric oxide but also support the hypothesis that an intrinsic immune defect may exist in cystic fibrosis. Key words: CFTR/chloride/lymphocytes/nitric oxide
Introduction Cystic fibrosis transmembrane conductance regulator (CFTR), the protein product of cystic fibrosis (CF) gene, functions as a cAMP-regulated Cl- channel in the apical membrane of secretory epithelial cells (reviewed in Riordan, 1993). While CFTR has been generally thought to be expressed in an epithelial cell specific fashion, evidence for CFTR expression and/or function as a cAMPregulated Cl- conductance has also been observed in other cell types, including lymphocytes (Chen et al., 1989; Bubien et al., 1990; Bremer et al., 1992; Krauss et al., 1992a,b; McDonald et al., 1992) and cardiac muscle cells (Levesque et al., 1992; Horowitz et al., 1993). The physiological relevance of CFTR expression in lymphocytes and other non-epithelial cells is unknown. C1transport has been generally implicated in membrane potential determination in resting lymphocytes, lymphocyte activation, CD8+ T cell-mediated cytotoxicity and in volume regulation (reviewed in Premack and Gardner, 1991). In all cells in which CFTR is expressed, it is regulated
by cAMP-dependent phosphorylation. It has recently been shown that CFTR can also be regulated by a cGMPdependent pathway in epithelial cells (Lin et al., 1992; Chao et al., 1994). This is the apparent mechanism by which heat-stable enterotoxin (STa), which activates a particulate guanylate cyclase receptor in the apical membrane of intestinal epithelial cells, induces the massive Cl- secretion underlying its pathogenicity of diarrhea (Chao et al., 1994). We asked whether a cGMP-dependent pathway of CFTR activation also exists in T lymphocytes. Cytosolic guanylate cyclase activity has been found in lymphocytes (Deviller et al., 1975; Cille et al., 1983), and a role for cGMP in lymphocyte proliferation and differentiation has long been postulated (Kaever and Resch, 1990). A potent activator of soluble guanylate cyclase is nitric oxide (NO) (Deguchi, 1977; Mittal and Murad, 1977; Bohme et al., 1984), which is produced in T lymphocytes from L-arginine by a cytokine-inducible nitric oxide synthase (NOS) (Kirk et al., 1990; Efron et al., 199 1; Vuorinen, 1992; Henry et al., 1993) or produced exogenously by macrophages when acting as cytotoxic effector cells (Henry et al., 1993). We hypothesized that NO, produced either endogenously or derived from exogenous sources, might activate lymphocyte CFTR via cGMP, analogous to the pathway in secretory epithelial cells.
Results L-arg, but not D-arg, induces chloride currents in normal T cells Tetanus toxoid-responsive, non-transformed CD4+ human T lymphocytes cloned from the peripheral blood of normal and CF-affected human subjects were studied by means of whole-cell patch clamp recordings under conditions designed to maximize Cl- currents and minimize the contribution of other ionic species. Initial studies were performed on the normal T cell clones. Cells were studied 2-3 days after treatment with interleukin-2 (IL-2; 20 U/ml). When excess L-arginine (250 ,uM to 1 mM), the substrate for NOS, was included in the recording pipette, a gradual increase in whole-cell Cl- current was seen in -84% (21/25) of cells, beginning within 5 min of establishing the whole-cell recording (Figure 1 A). The ratio of peak L-arginine-induced current at + 100 mV to the cell capacitance was 40.1 ± 7.7 pA/pF (n = 25, mean ± SEM), compared with a baseline (defined as within 1 min of establishing whole-cell recording) current of 2.8 ± 0.4 pA/pF. Current responses during voltage step protocols at 1 and 7 min of recording (Figure I B) displayed little voltage or time dependence, and the whole-cell current-voltage (I-V) relation during the peak L-arginine response was essentially linear (Figure 1C, solid line). The current recorded is carried predominantly by Cl- ions
2700 20) Oxford University Press
NO activates CFTR in human lymphocytes
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Fig. 1. Effect of L-arginine on the activation of whole-cell Cl- currents in normal human cloned T cells. (A) Representative continuous whole-cell patch clamp current recording at a holding potential of -70 mV in a cell perfused with 250 ,uM L-arg. The current begins -5 min after establishment of the whole-cell mode and increases over time. Intermittent voltage steps as described below interrupt the current trace at -70 mV. Initial recording gain (current/speed) is shown by the calibration bar. Unless otherwise indicated, all current recordings are at this gain. Recording gain for this trace was changed from 5 to 1 (i.e. the current calibration bar was increased from 400 to 2000 pA) where indicated by the asterisk. (B) Representative superimposed current traces obtained by intermittent voltage steps in 50 mV increments between -100 and +100 mV from a holding potential of -70 mV (protocol in inset) from a cell perfused with L-arg, recorded at baseline (upper traces; within 1 min of obtaining the whole-cell recording configuration) and during the response (lower traces; 7 min later). (C) Whole-cell I-V relation of the L-arg-induced Cl- current, derived in both normal (solid line) and low [Cl-] (dotted line) bath as indicated in the inset. (D) Representative continuous current recording from a T cell perfused with 250 gM D-arginine (D-arg). (E) Representative current recording from a T cell perfused with L-arg (250 tM) plus L-NA (100 ,uM). (F) Representative current recording from a T cell perfused with L-arg (250 giM) plus L-NMMA (100 ,uM). (G) Representative current recording from a T cell perfused with L-arg (250 gM) plus D-NMMA (100 ,uM). (H) Cumulative histogram of baseline and maximal outward Cl- currents at + 100 mV in T cells perfused with L-arg, D-arg or L-arg plus L-NA, L-NMMA and D-NMMA. Currents were normalized for cell size by dividing by cell capacitance, an index of cell surface area.
under these conditions because K+ and Na+ are absent from the recording solutions and Cs', used to replace K+, is known to block K+ channels. Furthermore, reduction of external [Cl-] shifted the reversal potential to +62 mV, close to the value of +65 mV predicted by the Nernst equation, assuming a perfect Cl--selective conductance (Figure IC, dotted line). 5-nitro-2(3-phenylpropylamino) benzoate (NPPB; 20 ,uM) (Fuller and Benos, 1992) blocked the Cl- current by 50-80% (data not shown). The properties of the C1- current, including linear I-V relation, voltageand time-independent gating and sensitivity to the C1channel blocker NPPB, are characteristic of the CFTR C1current. These properties differ from those of two other Cl- currents, the Ca2+-activated Cl- current and the hypotonicity-induced Cl- current, described in both lymphocytes (reviewed in Premack and Gardner, 1991; Lewis
et al., 1993) and epithelial cells (McCann et al., 1989; Worrell et al., 1989; Wagner et al., 1991). The effect of L-arginine is not simply that of a free amino acid. Replacement of L-arginine by D-arginine (250 ,uM), which is not a substrate of NOS, did not produce a response; whole-cell currents remained stable
for the duration of recording (>15 min) (Figure 1D). Furthermore, addition of the NOS inhibitor, N-nitro-Larginine (L-NA; 100 ,uM) or N-monomethyl L-arginine (L-NMMA; 100 ,uM) (Rees et al., 1990), to the pipette containing L-arginine (250 gM) abrogated the effect of the amino acid on current activation (Figure 1 E and F). By contrast, N-monomethyl D-arginine (D-NMMA; 100 ,uM), which is not an inhibitor of NOS, did not affect the L-arg response (Figure IG). Cumulative data consistent with the conclusion that L-arginine stimulates Cl-
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currents by serving as a substrate for NOS are shown in Figure lH.
Exogenous nitric oxide donors induce similar currents in normal T cells If L-arginine acts by serving as a substrate for NOS, we reasoned that exogenous NO should also activate Clcurrents in the normal human cloned T cells. Addition of the NO donor S-nitroso-N-acetylpenicillamine (SNAP; 100 gM) (Southam and Garthwaite, 1991) to the bath solution resulted in an activation of Cl- currents in 100% of cells (20/20), with a current onset time ranging from 30 s to 4 min. The response was stable in 50% (10/20) cells and transient in 50% (10/20) of cells, the latter having a current duration between 3 and 5 min (Figure 2A). The normalized current amplitude at + 100 mV increased to 201.1 ± 43.8 pA/pF (n = 20) at peak response compared with a baseline of 2.5 ± 0.3 pA/pF. The voltage- and time-independence of gating on repetitive voltage steps (Figure 2B), the linear I-V relation (Figure 2C, solid line) and the anion selectivity (Figure 2C, dotted line) were very similar to that seen for the L-arginineinduced current. Similar results were also obtained with photolytic release of 250 ,uM potassium nitrosylpentachlororuthenate (caged NO), which was included in the recording pipette (Figure 2D-F). Some 94% (17/18) of cells responded, among which 53% (9/17) gave transient response between 3 and 5 min. The current onset time after photolytic release of NO varied from 1 s to 2 min. We attribute the current onset delay to intervening enzymatic steps (see below). Variability in response delay may be exacerbated by decreasing light intensity with repetitive flashes over the life-time of the lamp and/or small shifts in focus among different flashes. Normalized current amplitude at +100 mV after flash increased to 145.8 ± 31.9 pA/pF (n = 18) compared with a baseline value of 1.9 ± 0.6 pA/pF. In comparable control experiments in which no caged NO was included in the pipette, cells were quiet for up to 15 min after flash (4.3 ± 2.8 pA/pF after flash versus 2.4 + 1.3 pA/pF before flash; n = 6). Cumulative data demonstrating Cl- current activation by NO donors are shown in Figure 2G.
cGMP is the second messenger of L-arg/NO action We asked next whether NO activated the Cl- current through the action of the second messenger, cGMP. When added to the bath, cpt-cGMP (a membrane permeant analog of cGMP, 1 mM) (Chao et al., 1994) activated Clcurrents with properties identical to those activated by L-arginine or NO donors (Figure 3A-C). All (12/12) cells responded. The peak cpt-cGMP response was 194.3 ± 46.5 pA/pF (n = 12) compared with a baseline of 2.8 ± 0.4 pA/pF The ability of cpt-cGMP to mimic the L-arginine and NO responses suggests that cGMP is the second messenger mediating the NO effect. Further substantiation was provided by experiments with LY83583 (200 ,uM), an inhibitor of soluble guanylate cyclase (Mulsch et al., 1988). When included in the pipette, LY83583 prevented Cl- current activation by excess intracellular L-arginine (250 ,uM) (Figure 3D) and by SNAP (100 ,uM) added to the external solution (Figure 3E). By contrast, inclusion of LY83583 in the pipette did not affect the activation of Cl- currents by cpt-cGMP (peak response 2702
156.8 + 52.3 pA/pF versus baseline 2.8 ± 0.7 pA/pF; n = 5) (Figure 3F). The ability of LY83583 to block the effect of L-arg and SNAP, combined with the ability of cpt-cGMP to bypass this inhibitory effect, suggests that NO activates the Cl- current by activation of soluble guanylate cyclase and generation of cGMP. Cumulative results supporting a role for guanylate cyclase and cGMP in NO activation of Cl- currents are shown in Figure 3G. In intestinal epithelial cells, cAMP-dependent protein kinase (PKA) appears to be the major mediator of cGMP effects on Cl- secretion (Chao et al., 1994). By binding to its receptor guanylate cyclase, STa induces massive intracellular elevations in cGMP, well above the Ka for both PKA (I ,uM) and cGMP-dependent protein kinase (PKG) (20 nM). In vitro experiments have confirmed that STa activates PKA (Forte et al., 1992). Furthermore, inhibitors of PKA, including Rp-8-Br-cAMPS and Walsh inhibitor, a potent and specific peptide inhibitor of PKA (Cheng et al., 1986; Chao et al., 1994), appear more effective at blocking the effect of STa than the PKG inhibitor Rp-8-Br-cGMPS (Chao et al., 1994). In an apparently similar fashion, Walsh inhibitor (1 uM) blocked the effect of L-arginine (250 ,M) on Cl- current activation in T cells (2.2 + 0.3 pA/pF versus baseline of 2.0 + 0.4 pA/pF; n = 5) while Rp-8-Br-cGMPS (40 jM) did not affect the L-arginine response (42.1 + 6.5 pA/pF versus baseline of 1.8 + 0.6 pA/pF; n = 9). This would suggest that, with respect to Cl- current activation, cGMP acts via PKA in lymphocytes.
CF-derived T cells fail to respond to L-arg or nitric oxide donors The characteristics of the NO-induced Cl- current, i.e. voltage- and time-independent gating, linear I-V relation and sensitivity to NPPB, are identical to those described for CFTR Cl- currents. To confirm that NO-induced Clcurrent flows through CFTR, we performed an identical set of experiments in CF-derived human T cell clones, in which an absence of cAMP-regulated Cl- conductance has been shown (unpublished data). No responses to L-arginine (250 jM to 1 mM; Figure 4A), SNAP (100,M; Figure 4B), photolytic release of caged NO (250 ,uM; Figure 4C) or cpt-cGMP (1 mM; Figure 4D) were seen in CF-derived T cells in multiple trials. By contrast, we observed a nearly immediate increase in Cl- current in response to the Ca2+ ionophore ionomycin (1 ,uM), even after the CF T cell failed to respond to SNAP (Figure 4E). lonomycin responses were seen in 61% (14/23) of cells, with a maximal response of 61.0 + 25.6 pA/pF compared with a baseline of 3.2 + 0.4 pA/pF (n = 23). As described previously, Ca2+-activated Cl- currents are characterized by time-dependent activation upon depolarization (Figure 4F) and an outwardly rectifying I-V relation (Figure 4G) (McCann et al., 1989; Worrell et al., 1989; Wagner et al., 1991). Cumulative data demonstrating the failure of CF T cells to respond to L-arginine, NO donors and membrane-permeant cGMP are shown in Figure 4H.
Discussion In summary, by use of NOS substrate and inhibitors, as well as NO donors, we show that NO activates Cl- current
NO activates CFTR in human lymphocytes
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Fig. 2. NO donors activate whole-cell C1- currents in normal human cloned T cells. (A) Continuous current recording of the response of a T cell to SNAP (100 gM), added where indicated by the vertical line. There was a transient (-3 min) increase in whole-cell current following SNAP addition. SNAP was first dissolved in DMSO as a 100 mM stock solution, and freshly diluted with bath solution to 10 mM stock solution during experimentation. (B) Current traces from a T cell at baseline (upper) and after response to 100 jM SNAP (lower). (C) Whole-cell I-V relation of the SNAP-induced current recorded under normal (solid line) and low [Cl-] (dotted line) bath. (D) Continuous current recording from a T cell perfused with 250 jM caged NO (included in the pipette solution), which was photolytically released by a 1 ms flash from a xenon arc flash lamp at the time indicated. (E) Current traces from a cell before (baseline) and after photolytic release of NO (flash). (F) Whole-cell I-V relation of the NO-induced current at normal (solid line) and low [Cl-] (dotted line) bath. (G) Cumulative histogram of baseline and maximal outward C1- currents at + 100 mV for cells exposed to SNAP, to photolytic release of NO and to a control flash (caged NO not included in the pipette).
in normal human cloned T lymphocytes. The inhibition of the NO effect by the guanylate cyclase inhibitor LY83583, as well as the mimicry of the NO effect by a membrane-permeant analog of cGMP, suggest that NO exerts its effect on lymphocyte Cl- currents through activation of cytosolic guanylate cyclase and generation of cGMP. Both the characteristics of the NO-activated C1current and the absence of response in CF-derived cloned
human T cells are consistent with the conclusion that NO activates CFTR in T cells.
Role of NO as a signal transducer in the immune system While it is generally accepted that NO plays an important role in murine host defense, with macrophage-derived NO functioning as a cytotoxic molecule for invading 2703
Y.Dong et al. 400
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Fig. 3. NO activates C1- currents via cytosolic guanylate cyclase and cGMP. (A) Representative continuous current trace from a normal T cell exposed to cpt-cGMP (I mM) added to the bath at the time indicated by the vertical line. Initial recording gain (current/speed) is shown by the calibration bar. Unless otherwise indicated, all current recordings are at this gain. Recording gain for this trace was changed from 5 to I (i. e. the current calibration bar was increased from 400 to 2000 pA) where indicated by the asterisk. (B) Representative current recordings at baseline (upper) and at the peak response to 1 mM cpt-cGMP (lower). (C) Whole-cell I-V relation of cpt-cGMP-induced Cl- current at normal (solid line) and low [C1-] (dotted line) bath. (D) Continuous current trace from a cell perfused with both L-arg (250 ,uM) and the guanylate cyclase inhibitor LY83583 (200 gM). (E) Continuous current trace from a cell perfused with LY83583 (200 ,uM) and subsequently exposed to SNAP (100 ,uM). (F) Continuous current trace from a cell perfused with LY83583 (200 ,uM) and subsequently exposed to 1 mM cpt-cGMP. Recording gain for this trace was changed from 5 to 1 (i. e. the current calibration bar was increased from 400 to 2000 pA) where indicated by the asterisk. (G) Cumulative histogram of baseline and maximal outward Cl- currents at + 100 mV for cells perfused with L-arg plus LY83583, cells perfused with LY83583 and then exposed to SNAP, cells perfused with LY83583 and then exposed to cpt-cGMP, and cells exposed to cpt-cGMP, respectively.
microorganisms and tumor cells (Moncada et al., 1991) and evidence for murine T cell-derived NO serving an autoregulatory role (Kirk et al., 1990; Taylor-Robinson et al., 1994), there is less evidence for NO having a role in human responses to infection. Evidence that exogenous NO activates membrane-associated tyrosine kinases and phosphatases and stimulates nuclear factor KB (NF-KcB) DNA binding in peripheral blood mononuclear cells (Lander et al., 1993a) indicates that NO may play a signal transducing role in the immune system. Endogenous NO production in B cells was recently demonstrated to play a role in latent and lytic EBV infection and B cell apoptosis (Mannick et al., 1994). These reported effects of NO in the human system were largely independent of cGMP but were influential on signaling pathways regulated through redox status (Lander et al., 1993b; Mannick et al., 1994). The evidence presented herein suggests that NO acts as a transducer molecule in IL-2-induced T lymphocyte clones by an alternative mechanism, activating soluble guanylate cyclase and producing cGMP, thereby initiating downstream events including the activation of CFTR. The 2704
effects of NO are presumably more than the activation of CFTR, which, in this case, serves as a reporter for other cGMP functions in lymphocytes. The roles of cyclic nucleotides in regulation of lymphocyte function have been extensively studied and reviewed (Plaut et al., 1980; Coffey and Hadden, 1985). The data suggest that cGMP promotes clonal proliferation and enhances the functions of mature, differentiated lymphocytes. Thus NO, through the activation of guanylate cyclase and the generation of cGMP, could enhance T lymphocyte cytotoxic function and proliferation, as well as serve as a direct cytotoxic molecule.
Functional significance of CFTR activation in T lymphocytes by NO The activation of CFTR Cl- currents by NO raises the question of the precise physiologic role played by these currents. In general, Cl- currents have been implicated in several T lymphocyte functions, including volume regulation (Grinstein et al., 1984; Lee et al., 1988), cytolysis by cytotoxic T cells (Gray and Russell, 1986;
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Fig. 4. NO fails to induce Cl1 current in CF-derived human T cell clones. (A) Continuous current trace from a CF T cell perfused with L-arg (250 gM). (B) Continuous current trace from a CF T cell exposed to SNAP (100 ,uM). (C) Continuous current trace from a T cell, perfused with caged-NO (250 ,uM) which was photolytically released by two sequential 1 ms flashes where indicated. (D) Continuous current trace from a CF T cell exposed to cpt-cGMP (1 mM). (E) Continuous current trace from a CF T cell which was initially exposed to SNAP (100 gM) at the time indicated. After no response was recorded over the 10-min period, ionomycin (1 ,uM) was added to the bath. lonomycin promptly induced an increase in the whole-cell Cl- current. (F) Representative whole-cell current traces from a CF T cell at baseline (upper) and at the peak response after exposure to ionomycin (1 ,uM). (G) Whole-cell I-V relation of ionomycin-induced Cl- current. Data are averaged over the final 80 ms of the pulse. (H) Cumulative histogram of the baseline and maximal outward Cl- currents at + 100 mV in CF T cells perfused with L-arg (250 ,uM) or exposed to SNAP (100 jM), to photolytic release of caged NO (250 jM), to cpt-cGMP (I mM), or to ionomycin (1 jM), respectively.
Prochazka et al., 1988) and Ca2+ influx associated with T cell activation (Rosoff et al., 1988). It is not known what contribution CFTR makes to each of these functions, but multiple studies confirm the transcription and expression of CFTR in lymphocytes (Chen et al., 1989; Bubien et al., 1990; Bremer et al., 1992; Krauss et al., 1992a,b; McDonald et al., 1992). The finding that two regulated second messenger pathways (cAMP and cGMP) converge on CFTR (Bubien et al., 1990; McDonald et al., 1993 and the data presented herein) suggests that CFTR may play a role in normal immune function and, therefore, that defective CFTR function may be manifested as defective immune responses in CF patients. Immune system
abnormalities, including depressed lymphoproliferative responses to Pseudomonas aeruginosa, lower percentages of helper T cells and decreased helper T cell function (Knutsen and Slavin, 1989; Lahat et al., 1989; Knutsen and Mueller, 1990; Knutsen et al., 1990), have been documented previously. The abnormality in regulation of CFTR function by two second messenger pathways
suggests that the immune abnormalities may be intrinsic rather than acquired. Intrinsic immune defects may in turn contribute to the chronic infection underlying the morbidity/mortality associated with CF lung disease.
Source of NO for the activation of CFTR in T lymphocytes The experimental data imply that either endogenously or exogenously derived NO activates CFTR in T lymphocytes. Published data also suggest that NO could serve both a paracrine and an autocrine function in lymphocytes in vivo. NO can be derived from a variety of cells that can be in the proximity of T lymphocytes, including macrophages, vascular endothelial cells and airway epithelial cells. Macrophages possess a soluble, NADPH- and Mg2+-dependent, inducible NOS (Lowenstein et al., 1992, 1993); NO produced by macrophages mediates the ability to kill or inhibit the growth of tumor cells, bacteria, fungi and parasites (Moncada et al., 1991; Lowenstein and Snyder, 1992). Vascular endothelial cells contain a Ca2+-
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regulated, constitutively produced NOS (Janssen et al., 1992; Lamas et al., 1992; Sessa et al., 1992); NO produced by endothelial cells mediates vascular smooth muscle relaxation and inhibits platelet aggregation (reviewed in Moncada and Higgs, 1993). Bronchial epithelium contains a neuronal type NOS, which is Ca2+/calmodulin-regulated and constitutively produced (Kobzik et al., 1993); NO produced in airway epithelium is thought to play a role in bronchial smooth muscle relaxation and ventilationperfusion matching (Gaston et al., 1994). Thus several tissues could serve as a ready source for paracrine regulation of T lymphocytes by NO. In addition to these exogenous sources, T lymphocytes may also produce NO. T cells have been reported to have an IL-2-inducible NOS (Kirk et al., 1990; Efron et al., 1991; Vuorinen, 1992; Henry et al., 1993), and NO produced by IL-2-stimulated T cells has been shown to inhibit platelet aggregation in vitro (Kirk et al., 1990).
Speculation of the regulation of CFTR by NO in other cell types NO regulation of CFTR may not be restricted to T lymphocytes. CFTR expression is most prevalent in secretory epithelial cells, which line the airways, pancreatic ducts, intestinal tract and sweat glands. NO production has been reported in and around several secretory epithelial cell types. In the bronchial airway, NO is produced by epithelial cells and adventitial nerves (Schmidt et al., 1992a; Kobzik et al., 1993; Jorens et al., 1993). In intestinal epithelial cells, D-glucose stimulates L-argininedependent NO formation and is reported to be involved in ion secretion (Schmidt and Walter, 1994). In the pancreas, L-arginine-derived NO is reported to induce insulin secretion by the ,-cells (Schmidt et al., 1992b). In addition, CFTR is expressed in cardiac myocytes, which are innervated by nitroxergic neurons mediating negative inotropy (Balligand et al., 1993). The co-existence of CFTR with sites of NO production in several cell types suggests that this process may not be unique to T lymphocytes and may therefore play a role in a variety of functions such as ion secretion from epithelial cells or membrane potential determination and contractility of myocardial cells.
Materials and methods Reagents L-arg, D-arg, D-NMMA, L-NMMA and L-NA were purchased from Sigma, St Louis, MO. SNAP was obtained from Research Biochemicals Incorporated, Natick, MA. Caged NO was obtained from Molecular Probe, Eugene, OR. cpt-cGMP and Rp-8-Br-cGMPS were purchased from BioLog, Germany. LY83583 was obtained from CalBiochem, La Jolla, CA.
Cells CF patients were diagnosed by the sweat chloride test (Gibson and Cooke, 1959) and genotype mapping (Riordan et al., 1989). Peripheral blood T lymphocytes were isolated from either normal healthy volunteers or CF-affected patients by standard Ficoll-Hypaque density gradients and cultured for 7-10 days in the presence of tetanus toxoid (Connaught Laboratory) to generate tetanus toxoid-specific T cell lines (Fathman and Fitch, 1982). Cells were maintained in 5% CO2 in RPMI 1640 cell culture medium (Applied Scientific), supplemented with 5% heatinactivated human AB serum (Irvine Scientific), penicillin (100 U/mI), streptomycin (100 gg/ml) and glutamine (2 mM). Clones were generated by limiting dilution (one cell per three wells) culture in the presence of
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a feeder cell mixture of antigen, leukagglutnin A (1 ,ug/ml), irradiated autologous and allogenic peripheral blood mononuclear cells (3000 rads) or EBV-B cells (5000 rads). Recombinant IL-2 (20 U/ml; Genzyme) was added on day 4 and maintained through a full growth cycle for clonal expansion (10-14 days). At the end of the growth cycle, cells were re-stimulated following the same protocol described above.
Electrophysiology Standard whole-cell patch clamp recording techniques were used (Hamill et al., 1981). Cells were placed in an acrylic/polystyrene perfusion chamber mounted on the stage of an inverted microscope (Nikon, Diaphot). Experiments were performed at 30°C. Extracellular (bath) solution had the following composition: 170 nM Tris-CI, I mM MgCl2, 2.5 mM CaC12, 10 mM HEPES and 15 mM glucose (pH 7.4, 325 mosm/kg). Reduction of [Cl-] in the external solution was achieved by replacement of Tris-CI with Tris-aspartate (external [Cl-] 11 mM). The intracellular (pipette) solution had the following composition: 140 mM CsCl, 2 mM MgCl2, 2 mM Mg-ATP, 0.5 mM Li-ATP, I mM EGTA, 5 mM HEPES and 10 mM glucose (pH 7.35, 300 mosm/kg). The difference between osmolarities of the bath and pipette solutions was required to prevent hypotonicity-induced Cl- currents (Worrell et al., 1989; Wagner et al., 1991). Micropipettes had a tip resistance of2-5 MQ when filled with pipette solution. Whole-cell patch clamp recordings were made using a model IC Axopatch amplifier (Axon Instruments). Signals were filtered at 1 kHz and stored on floppy disks. Data were analyzed by means of pClamp, version 5.5 (Axon Instruments). Sample sizes are presented as n = number of whole-cell patches. Flash photolysis of caged NO was achieved by a 1 ms flash from a high-pressure xenon arc flash lamp (G.Rapp, Heidelberg, Germany) as described previously (McDonald et al., 1993). Wavelengths
