Activation of CD38 by interleukin-8 signaling regulates intracellular Ca2+ level and motility of lymphokine-activated killer cells.

CD38 is an ADP-ribosyl cyclase, producing a potent Ca(2+) mobilizer cyclic ADP-ribose (cADPR). In this study, we have investigated a role of CD38 and its regulation through interleukin-8 (IL8) signaling in lymphokine-activated killer (LAK) cells. Incubation of LAK cells with IL8 resulted in an increase of cellular cADPR level and a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)), which was sustained for a long period of time (>10 min). Preincubation of an antagonistic cADPR analog, 8-Br-cADPR (8-bromo-cyclic adenosine diphosphate ribose), abolished the sustained Ca(2+) signal only but not the initial Ca(2+) rise. An inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist blocked both Ca(2+) signals. Interestingly, the sustained Ca(2+) rise was not observed in the absence of extracellular Ca(2+). Functional CD38-null (CD38(-)) LAK cells showed the initial rapid increase of [Ca(2+)](i) but not the sustained Ca(2+) rise in response to IL8 treatment. An increase of cellular cADPR level by cGMP analog, 8-pCPT-cGMP (8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate), but not cAMP analog or phorbol 12-myristate 13-acetate was observed. IL8 treatment resulted in the increase of cGMP level that was inhibited by the IP(3) receptor blocker but not a protein kinase C inhibitor. cGMP-mediated Ca(2+) rise was blocked by 8-Br-cADPR. In addition, IL8-mediated LAK cell migration was inhibited by 8-Br-cADPR and a protein kinase G inhibitor. Consistent with these observations, IL8-induced migration of CD38(-) LAK cells was not observed. However, direct application of cADPR or 8-pCPT-cGMP stimulated migration of CD38(-) cells. These results demonstrate that CD38 is stimulated by sequential activation of IL8 receptor, IP(3)-mediated Ca(2+) rise, and cGMP/protein kinase G and that CD38 plays an essential role in IL8-induced migration of LAK cells.

A type II transmembrane protein CD38, originally known as an activation antigen, displays ADP-ribosyl cyclase (ADPRcyclase) 1 and cyclic ADP-ribose hydrolase (cADPR-hydrolase) activities (1,2). These two enzyme activities are involved in the conversion of ␤-nicotinamide adenine dinucleotide (␤-NAD ϩ ) first to cyclic ADP-ribose (cADPR) and then to ADP-ribose (ADPR) (3)(4)(5). CD38 is also ADP-ribosylated by ecto-ADP-ribosyltransferase in the presence of exogenous ␤-NAD ϩ (6). This modification results in inactivation of the enzyme activity. The metabolite cADPR is known to induce Ca 2ϩ release from intracellular stores by acting on ryanodine receptor and/or Ca 2ϩ influx through plasma membrane Ca 2ϩ channels in a variety of cells (7)(8)(9)(10)(11). Several studies have indicated that cADPR synthesis by CD38 is stimulated through cell surface heterotrimeric G-protein-coupled receptor signaling. The receptors include ␤-adrenergic receptor in cardiac myocytes (12) and artery smooth muscle cells (13), angiotensin II receptor in cardiac myocytes (14), muscarinic receptor in neuroblastoma NG-108 (15), and pancreatic acinar cells (16). The activation of ADPRcyclase by cGMP in Aplysia califonica has been reported (17), and cAMP-dependent activation of the enzyme is also observed in artery smooth muscle cells (13). However, the molecular basis of the activation of CD38 and/or ADPR-cyclases has not been clearly defined.
A previous report has indicated that a peptide cytokine interleukin (IL) 8 signaling may utilize cADPR to mobilize [Ca 2ϩ ] i in IL2-activated natural killer cells (18). IL8, which belongs to the CXC superfamily of chemokines, plays an important role in the motility of various cells such as neutrophils and T cells (19,20) and also induces angiogenesis and other effects associated with proinflammatory responses (21)(22)(23). The IL8 receptor (IL8R) is made of seven transmembrane proteins and couples with G i and stimulates the production of inositol 1,4,5-trisphosphate (IP 3 ) through the activation of phospholipase C (PLC)-␤2 (24,25). There is a report that the IL8Rs present in natural killer cells and lymphokine-activated killer (LAK) cells may induce cADPR synthesis through the G s -involved signaling pathway (26). CD38 expression in natural killer and LAK cells is also observed (27,28). However, the role of CD38 including cADPR in LAK cell functions and the activation pathways of CD38 remain elusive.
In this study, we have investigated IL8-mediated regulation of CD38 by determining intracellular Ca 2ϩ changes and motility of LAK cells. The results indicate that CD38 is activated via cGMP/protein kinase G (PKG) that is activated by IL8 and that CD38 plays a critical role in IL8-mediated Ca 2ϩ signal and migration of LAK cells.

EXPERIMENTAL PROCEDURES
Reagents and Antibodies-Antibodies were obtained as follows: antihuman CD38 monoclonal antibody from BD Biosciences; anti-IL8R monoclonal antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-PLC-␤2 monoclonal antibody from P. G. Suh at POSTECH (Pohang, Korea); anti-IP 3 receptor (IP 3 R) generously provided by S. H. Kim (Inha University, Incheon, Korea); anti-ryanodine receptor (RyR) common mouse monoclonal antibody from Calbiochem. Ficoll-Hypaque and Percoll were obtained from Amersham Biosciences. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG was purchased from Advanced Biochemicals Inc. (Jeonju, Korea). Human recombinant IL2 was purchased from Chiron BV (Amsterdam, Netherlands). Human recombinant IL8, human AB serum, and all other reagents were obtained from Sigma. RPM1 1640 was from Invitrogen. 125 I-cGMP radioimmunoassay kit was from PerkinElmer Life Sciences.
Preparation of LAK Cells-LAK cells were prepared as described previously (29,30). Briefly, blood obtained from healthy volunteers was layered over Ficoll-Hypaque and centrifuged at 700 ϫ g for 30 min to remove red blood cells. The red blood cells removed cell preparations were incubated on a nylon-wool column at 37°C for 1 h in a 5% CO 2 incubator to remove B lymphocytes and macrophages. Nylon-wool nonadherent cells were collected and further separated by a Percoll density gradient centrifugation. Four layers of Percoll were used: 37, 44, 52, and 60%. After centrifugation at 700 ϫ g for 20 min, cells of the 52% Percoll layer were collected, washed with serum-free RPMI 1640, and incubated at 2 ϫ 10 6 cells/ml density in a culture media containing 3000 IU/ml IL2 in a 5% CO 2 incubator at 37°C. The culture medium used is RPMI 1640 supplemented with 10% human AB serum, 0.25 g/ml amphotericin B, 50 g/ml gentamycin, 10 units/ml penicillin G, 100 g/ml streptomycin, 1 mM L-glutamine, 1% nonessential amino acids, and 50 M 2-mercaptoehanol. After incubation for 24 h, the floating cells were removed, and the adherent cells were cultured in the same culture media containing 1500 IU/ml IL2. LAK cells induced by IL2 for 10 days were used throughout the study, otherwise indicated in the figure legends. Induction of LAK cells was ensured by determining ADPR-cyclase activity as detailed below.
Synthesis of cADPR-cADPR was synthesized using ADPR-cyclase purified from A. califonica ovotestes as described previously (31). The nucleotide was purified by high performance liquid chromatography (HPLC) using AG MP-1 resin (Bio-Rad). The nucleotide was eluted with a nonlinear gradient of 150 mM trifluoroacetic acid and water. Purified cADPR was dried using a SpeedVac concentrator. The purity of cADPR used in the study was ϳ97% as determined by HPLC.
␤-NAD ϩ -dependent Inactivation of CD38 -LAK cells were incubated with 100 M ␤-NAD ϩ for 6 h according to the method described previ-ously (6), and after incubation, the cells were washed with phosphatebuffered saline and used for the study.
Measurement of cADPR i -The level of [cADPR] i was measured using a cyclic enzymatic assay as described previously (33). Briefly, LAK cells were treated with 0.5 ml of 0.6 M perchloric acid under sonication. Precipitates were removed by centrifugation at 20,000 ϫ g for 10 min. Perchloric acid was removed by mixing the aqueous sample with a solution containing 3 volumes of 1,1,2-trichlorotrifluoroethane to 1 volume of tri-n-octlyamine. After centrifugation for 10 min at 1500 ϫ g, the aqueous layer was collected and neutralized with 20 mM sodium phosphate (pH 8). To remove all contaminating nucleotides, the samples were incubated with the following hydrolytic enzymes overnight at 37°C: 0.44 unit/ml nucleotide pyrophosphatase, 12.5 units/ml alkaline phosphatase, 0.0625 unit/ml NADase, and 2.5 mM MgCl 2 in 20 mM sodium phosphate buffer (pH 8.0). Enzymes were removed by filtration using Centricon-3 filters. To convert cADPR to NAD ϩ , the samples (0.1 ml/tube) were incubated with 50 l of a cycling reagent containing 0.3 g/ml Aplysia ADPR-cyclase, 30 mM nicotinamide, and 100 mM sodium phosphate (pH 8) at room temperature for 30 min. The samples were further incubated with the cycling reagent (0.1 ml) containing 2% ethanol, 100 g/ml alcohol dehydrogenase, 20 M resazurin, 10 g/ml diaphorase, 10 M riboflavin 5Ј-phosphate, 10 mM nicotinamide, 0.1 mg/ml BSA, and 100 mM sodium phosphate (pH 8.0) for 2 h at room temperature. An increase in the resorufin fluorescence was measured at 544 nm excitation and 590 nm emission using a fluorescence plate reader (Molecular Devices Corp., Spectra-Max GEMINI). Various known concentrations of cADPR were also included in the cycling reaction to generate a standard curve.
Measurement of Intracellular cGMP Level-Levels of cGMP were determined by a 125 I-cGMP radioimmunoassay kit according to the manufacturer's protocol. LAK cells were preincubated with 0.5 mM isobutylmethylxanthine and phosphodiesterase inhibitor and then challenged with various reagents as detailed in the figure legends. After incubation for 30 min, cells were treated with equal volume of 12% trichloroacetic acid. To determine cGMP extraction efficacy, [ 3 H]cGMP (1500 cpm) was added. After centrifugation at 20,000 ϫ g at 4°C for 10 min, supernatants were collected and extracted four times with a 5-ml portion of water-saturated diethyl ether. The water part was collected, dried using SpeedVac, and dissolved in 200 l of 50 mM sodium acetate buffer (pH 6.2). Prior to performing radioimmunoassay, the sample (100 l) was acetylated using acetic anhydride in the presence of triethylamine. A standard curve of acetylated cGMP was also prepared as described in the manufacturer's protocol.  (34). The emitted fluorescence at 530 nm was collected using a photomultiplier. One image every 6 s for 10 min was scanned using confocal microscope (Nikon, Japan). For the calculation of [Ca 2ϩ ] i , the method of Tsien et al. (35) was used with the following equation:

Measurement of [Ca
where K d is 450 nM for Fluo-3 and F is the observed fluorescence levels. Each tracing was calibrated for the maximal intensity (F max ) by the addition of ionomycin (8 M) and for the minimal intensity (F min ) by the addition of EGTA 50 mM at the end of each measurement.
Determination of Cell Migration-Cell migration was determined as described previously (28). In brief, cells were washed with RPMI 1640, scrapped using a policeman, and washed with RPM1 1640. Transwells (Costar Corning, Corning, NY) with 8-m pore size polycarbonate filters 6.5 mm in diameter were used. Lower chambers contained 500 l of RPMI 1640 containing 1% BSA. LAK cells (4 ϫ 10 5 ) in 100 l RPM1 1640 containing various agents (specified in the figure legends) were placed in the upper chamber. The chambers were incubated in a 5% CO 2 incubator at 37°C for 2 h. The filters were removed, fixed with ice-cold 100% methanol, and stained with 15% Wright-Giemsa stain for 7 min. The cells were counted under a phase-contrast microscope.
Statistical Analysis-Data represent means Ϯ S.E. of the mean (S.E.) of at least three separate experiments. Statistical analysis was performed using Student's t test. A value of p Ͻ 0.05 was considered significant.

Induction of CD38 Expression in LAK Cells-Firstly,
we assessed expression levels of CD38 along with the other related signaling molecules during induction of LAK cells by IL2 treatment. Expression of CD38 was gradually increased in a timedependent manner and highly induced at 7-10 days (Fig. 1A). Expression of RyR, a putative receptor for cADPR, was also highly induced at 7-10 days. On the other hand, expression of IL8R, IP 3 R, and PLC-␤2 was observed in the freshly isolated T cells and was not influenced by IL2 treatment (data not shown). Expression of CD38 was ascertained by measuring ADPR-cyclase activity using NGD ϩ , which is converted to cGDPR only by the enzyme (Fig. 1B). Consistent with the above observations, the production of cGDPR in cell lysates was increased in a time-dependent manner. Next, we examined whether IL8 stimulates CD38 in LAK cells induced by treatment with IL2 for 10 days. As shown in Fig. 1C, [cADPR] i in LAK cells was increased significantly by the treatment with IL8. These results show that expression of CD38 and RyR along with transformation of T cells to LAK cells are induced by IL2 treatment and that IL8 signaling may activate CD38.
IL8-mediated Elevation of [Ca 2ϩ ] i Involves Activation of CD38 -On the basis of the above observation that IL8 treat-ment increases [cADPR] i , the molecular basis of CD38 activation by IL8 in LAK cells was examined by determining IL8mediated changes in [Ca 2ϩ ] i under various conditions. The addition of IL8 to the cells resulted in a rapid increase in [Ca 2ϩ ] i , and the increased [Ca 2ϩ ] i levels were sustained for more than 10 min (Fig. 2A). The pretreatment of an antagonistic cADPR analog, 8-Br-cADPR, abolished the IL8-mediated sustained Ca 2ϩ signal but not the initial rapid increase (Fig.  2B). Interestingly, xestospongin C, an IP 3 receptor antagonist, completely abolished both Ca 2ϩ signals (Fig. 2C). In contrast, calphostin C, a protein kinase C inhibitor, had no effect on the IL8-mediated increase of [Ca 2ϩ ] i (Fig. 2D). To examine whether the sustained Ca 2ϩ increase is due to Ca 2ϩ release from the intracellular store or Ca 2ϩ influx, the IL8-mediated increase of [Ca 2ϩ ] i was determined in the presence of EGTA. The sustained Ca 2ϩ rise was not observed while the initial Ca 2ϩ increase was present (Fig. 2E). The effects of various agents on the initial and sustained Ca 2ϩ increases were summarized in Fig. 2, F and G, respectively. These results indicate that cADPR is responsible for IL8-mediated sustained Ca 2ϩ influx that is dependent on the IP 3 -mediated initial Ca 2ϩ rise in the LAK cells.
Inactivation of CD38 by ADP-ribosylation Abolishes the Sustained Rise of [Ca 2ϩ ] i Induced by IL8 -We have previously demonstrated that CD38 in the activated T cells is ADP-ribosylated in the presence of exogenous ␤-NAD ϩ , resulting in a loss of enzyme activity (6). To further elucidate the role of CD38/cADPR in IL8-induced Ca 2ϩ signaling, functional CD38null (CD38 Ϫ ) LAK cells were prepared by treatment with ␤-NAD ϩ . As shown in Fig. 3A, ADPR-cyclase activities, which were observed in the lysates and CD38 immunoprecipitates prepared from the control LAK cells, were completely eradicated by the incubation with ␤-NAD ϩ . These results indicated that CD38 represents the only ADPR-cyclase in LAK cells but also indicate that CD38 is inactivated in the presence of ␤-NAD ϩ , consistent with previous observations (6). When IL8-mediated Ca 2ϩ signal in CD38 Ϫ LAK cells was compared with the control LAK cells (Fig. 3B), CD38 Ϫ LAK cells showed only the initial rise of [Ca 2ϩ ] i but not the sustained rise of [Ca 2ϩ ] i by the treatment with IL8 (Fig. 3C). To ensure that the sustained Ca 2ϩ signal is mediated by cADPR, changes of [Ca 2ϩ ] i in the CD38 Ϫ LAK cells were determined in the presence of cADPR. Indeed, cADPR was able to induce the sustained Ca 2ϩ signal (Fig. 3D). As summarized in Fig. 3, E and F, the sustained Ca 2ϩ increase is due to the activation of CD38/cADPR through IL8 signaling.
CD38 Is Activated by cGMP Produced by IL8/Ca 2ϩ Signaling-It has been demonstrated that ADPR-cyclases including CD38 are probably activated by cAMP or cGMP (13,17). Stimulation of IL8R produces two second messengers, IP 3 and diacylglycerol. There is a report that IL8R may couple with G s (26). To elucidate activation mechanism of CD38 by IL8 signaling, we determined [cADPR] i in LAK cells treated with various agonistic agents. As presented in Fig. 4A, a membrane-permeable cGMP analog, 8-pCPT-cGMP, significantly increased the level of [cADPR] i that was comparable with that induced by IL8. Moreover, a membrane-permeable PKG inhibitor, Rp-8-pCPT-cGMPS, blocked IL8-induced cADPR formation. On the other hand, phorbol 12-myristate 13-acetate or the cAMP analog, 8-CPT-cAMP, or Ca 2ϩ ionophore, ionomycin, was not able to increase [cADPR] i . We further examined whether cGMP is produced by stimulation of IL8R. The treatment of IL8 indeed increased the intracellular cGMP level, and the IL8-induced cGMP increase was completely blocked by xestospongin C but not by calphostin C (Fig. 4B). Supporting the result that ionomycin had no effect on cADPR formation, cGMP level was also FIG. 1. Expression of CD38 and RyR during induction of LAK cells. Isolated T lymphocytes were treated with IL2 for 10 days. During induction of LAK cells, expression of CD38 and RyR was determined. A, CD38 and RyR are expressed during induction of LAK cell by IL2. A typical Western blot of CD38 and RyR expression during induction of LAK cells is shown. Cell lysate (20 g) was subjected to immunoblotting using CD38 and RyR antibodies. B, increase of ADPR activity during LAK cell induction. ADPR cyclase activity in cell lysates (10 g) prepared from IL2 treated cells was determined using 200 M NGD ϩ as a substrate. NA, no activity found. C, [cADPR] i is increased in response to IL8 treatment. LAK cells were incubated with IL8 (10 pM) for 1.5 min. Formation of cADPR was determined as described under "Experimental Procedures." The data Ϯ S.E. from three independent experiments are shown. **, p Ͻ 0.005. not increased by the ionomycin treatment. The observations that initial Ca 2ϩ rise proceeded to increase the sustained Ca 2ϩ signal (Fig. 2, A and B) indicated that cGMP increase induced by IL8 may proceed to form cADPR. The time course of cGMP and cADPR formation induced by IL8 was evaluated. The results showed that levels of cGMP and cADPR were increased in a time-dependent manner, reaching maximal levels at ϳ60 and 90 s, respectively (Fig. 4C). The formation of cGMP was rapidly reduced, whereas the formation of cADPR was slowly decreased. Supporting these results, a cGMP analog, 8-pCPT-cGMP, generated the sustained increase of [Ca 2ϩ ] i (Fig. 4D). The cGMP analog-mediated increase of [Ca 2ϩ ] i was completely blocked by pretreatment with 8-Br-cADPR (Fig. 4E) but not by xestospongin C (Fig. 4F). Moreover, Rp-8-pCPT-cGMPS blocked IL8-induced sustained Ca 2ϩ rise but not the initial Ca 2ϩ rise (Fig. 4G). Differences in [Ca 2ϩ ] i during initial and sustained increases of [Ca 2ϩ ] i were summarized in Fig. 4, H and I, respectively. These findings indicate that cGMP is involved in the activation of CD38 in LAK cells and that IL8induced cGMP formation is due to IP 3 -mediated Ca 2ϩ rise. In addition, we also assessed whether phosphorylation of CD38 via cGMP/PKG during IL8 treatment occurred. Phosphorylation of CD38 was not observed. The increased production of cGDPR by CD38 isolated from LAK cells by immunoprecipitation with CD38 antibody was not observed in the presence of cGMP, PKG, or both together or Ca 2ϩ (data not shown).
cGMP/PKG/cADPR Signaling System Is Essential for IL8induced Migratory Activity of LAK Cells-It is well known that the activation of IL8R induces migration of various cell types (19,20). To examine whether CD38/cADPR plays any role in IL8-mediated cell migration, we evaluated the effect of cADPR on the migratory activity of LAK cells. As presented in Fig. 5A 8-Br-cADPR. In addition, we also examined the effects of Ca 2ϩ on LAK cell migration (Fig. 5A). IL8-induced cell migration was not observed in the absence of extracellular Ca 2ϩ , and the increase of [Ca 2ϩ ] i by ionomycin did not induce cell migration.
The above observations that CD38/cADPR is involved in LAK cell migration were further examined using CD38 Ϫ LAK cells. IL8-meidated migration of CD38 Ϫ LAK cells was not observed (Fig. 5B). However, the treatment of CD38 Ϫ LAK cells with exogenous cADPR stimulated cell migration similar to the control LAK cells induced by IL8. To further support the observation that cGMP is able to activate CD38, thus resulting in an increase of cADPR production, cGMP-mediated LAK cell migration was examined. An agonistic membrane-permeable cGMP analog, 8-pCPT-cGMP, stimulated the migratory activity of LAK cells independent of the activation of IL8R (Fig. 5C). The pretreatment of PKG inhibitor, Rp-8-pCPT-cGMPS, completely blocked IL8-induced migration of LAK cells. These re-sults indicate that CD38/cADPR plays an important role in IL8-mediated migration of LAK cells.

DISCUSSION
CD38 is a bifunctional enzyme having ADPR-cyclase and hydrolase activity that produces and hydrolyzes cADPR, which is a powerful and universal Ca 2ϩ -mobilizing second messenger. Studies have proposed that CD38, including ADPR-cyclases, is activated by G-protein-coupled receptor. However, the regulation pathway(s) of CD38 by G-protein-coupled receptor remains unclear. In this study, we for the first time demonstrate that CD38 induced in LAK cells is stimulated by cGMP/PKG that is generated upon the activation of IL8R. Our results have also revealed that a CD38 metabolite, cADPR, plays an essential role in regulation of [Ca 2ϩ ] i and migratory activity of LAK cells.
The regulation pathway of CD38 in IL8 signaling is summarized in Fig. 6. IL8R couples with G i and stimulates PLC-␤2, producing two second messengers, IP 3 and diacylglycerol (24,25). The IL8 treatment of LAK cells exhibits a rapid rise of [Ca 2ϩ ] i that sustains for a long period of time (Ͼ10 min). Our results show that the initial Ca 2ϩ signal is mediated by IP 3 and that the IL8-mediated sustained Ca 2ϩ signal is due to the activation of CD38, resulting in an increase of [cADPR] i . Thus, the pretreatment of cells with 8-Br-cADPR, an antagonistic analog of cADPR, displays only the initial rise of [Ca 2ϩ ] i . An IP 3 R blocker, xestospongin C, abolishes completely the IL8mediated elevation of the initial and sustained [Ca 2ϩ ] i , indicating that the activation of CD38 requires the IP 3 -mediated increase of [Ca 2ϩ ] i . However, in contrast to IL8-induced forma-   (10). Together, it appears that CD38 in LAK cells, including T cells, is not activated unspecifically by increased [Ca 2ϩ ] i . Our results also indicate that cADPR induces Ca 2ϩ influx in LAK cells. The depletion of extracellular Ca 2ϩ using EGTA generates the initial Ca 2ϩ rise but not the sustained Ca 2ϩ signal in response to IL8. CD38/cADPR-mediated Ca 2ϩ entry has been observed with neutrophils (11) and intact human T cells (36). A recent study has reported that type 3 RyR plays an essential role in the sustained Ca 2ϩ response in T cells (37). Although the molecular mechanism by which cADPRmediated sustained Ca 2ϩ rise remains to be clarified, our results strongly suggest that cADPR produced by CD38 is a Ca 2ϩ -mobilizing second messenger and mediates the sustained phase of Ca 2ϩ signal by means of Ca 2ϩ influx in LAK cells.
Studies have indicated that cAMP activates the production of cADPR in artery smooth muscle cell (13) and that cGMP is the activator of A. califonica ADPR-cyclase (17). Our results suggest that the activation of CD38 expressed in LAK cells is mediated by PKG activation via cGMP generated by IL8 signaling. Thus, the treatment of various agents shows that cGMP increases [cADPR] i in LAK cells. Consistent with these results, cGMP induces the sustained rise of [Ca 2ϩ ] i , and a PKG blocker, Rp-8-pCPT-cGMPS, abolishes the sustained Ca 2ϩ signal but not the initial Ca 2ϩ signal in response to IL8. More importantly, IL8-induced increase of cGMP precedes the production of cADPR. These results demonstrate that the activation of IL8R increases cGMP production in LAK cells and that cGMP/ PKG regulates CD38 activity. However, it appears that cGMP/ PKG does not directly act on the enzyme; neither PKG-mediated phosphorylation of CD38 nor cGMP-mediated activation of isolated CD38 using the CD38 antibody is observed.
Our results have indicated that CD38/cADPR plays a critical role in the IL8-mediated migration of LAK cells. Thus, IL8mediated cell migration is blocked by an antagonistic cADPR analog, 8-Br-cADPR, and by inactivation of CD38 with ␤-NAD ϩ . Moreover, cADPR alone is able to induce LAK cell migration. Consistent with the observations that cGMP/PKG  6. Schematic presentation of IL8-mediated CD38 activation in LAK cell. Ligation of IL8R receptor stimulates PLC-␤2 via G i , resulting in the production of IP 3 and diacylglycerol (24,25). IP 3 binding to the receptor releases Ca 2ϩ from the intracellular Ca 2ϩ stores. The IP 3 -mediated increase of the intracellular Ca 2ϩ level results in the elevation of the cGMP level via guanylyl cyclase (GC), thus activating PKG. cGMP/PKG-mediated signaling stimulates intracellular cADPR production via CD38. cADPR-mediated Ca 2ϩ influx is requisite for LAK cell migration. cADPR may act on RyR present on Ca 2ϩ store to regulate the sustained Ca 2ϩ rise in LAK cells since type 3 RyR in T cells regulates the sustained Ca 2ϩ response (37). signaling activates CD38, the pretreatment of the PKG inhibitor blocks IL8-mediated migration of LAK cells. We have also found that IL8-induced LAK cell migration is strictly controlled by Ca 2ϩ influx; in the absence of extracellular Ca 2ϩ , IL8-induced migration of LAK cells is not observed, and nonphysiological increase of [Ca 2ϩ ] i with ionomycin does not induce cell migration. A previous study has also indicated that dendritic cell trafficking depends on Ca 2ϩ influx induced by CD38/ cADPR (38). Studies have demonstrated that the biological role of CD38/cADPR in LAK cells as well as natural killer cells isolated is to trigger lytic and secretory responses (27). This cytotoxic role of CD38 has been observed by direct ligation of CD38 with an agonist antibody IB4 (39). The main signaling event in the cytotoxicity seems to be an involvement of protein tyrosine kinase(s) signaling via binding of the agonistic antibody IB4 to CD38 (39,40). Taken together, CD38 plays important roles in migration and cytotoxicity of LAK cells, including natural killer cells. The present study shows the regulationsignaling pathway of CD38 through the sequential coupling of IL8R, Ca 2ϩ and cGMP/PKG and describes the biological role of CD38 in this signaling pathway, which is involvement in IL8induced cell migration through the generation of cADPR/sustained Ca 2ϩ elevation.