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*
Department of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, TN 38105;
First Department of Internal Medicine, Kagawa Medical University, Kagawa, Japan; and
University of Tennessee College of Medicine, Memphis, TN 38163
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The relation between the enzymatic activity and the signaling properties of CD38 is unclear. Based on the crystal structure of Aplysia ADPR cyclase, an enzyme homologous to CD38, Prasad et al. proposed a model of CD38 as a dimer that encloses a cavity supporting the ectoenzymatic activity 15 . These authors speculated that conformational changes associated with the enzymatic activity could be transmitted to the cytoplasmic domain of CD38, resulting in protein tyrosine kinase activity. The molecular mechanisms linking CD38 ligation with its effects in immature B lymphoid cells are also unclear. In mature B and T lymphocytes CD38 signaling depends on the integrity of the B and T cell Ag receptors, respectively 16, 17, 18 , which are not expressed on the surface of immature B cells.
In the present study we used Ba/F3 murine pro-B cells transfected with a cDNA encoding human CD38 to further clarify the signaling mechanism mediated by CD38 in immature lymphoid cells. We found that ligation of human CD38 ectopically expressed in murine pro-B cells results in signaling events that recapitulate those seen in their human counterparts. In addition, ligation of CD38 in the murine transfectants caused marked homotypic aggregation followed by cell death. Identical results were obtained in cells transfected with a cDNA encoding a mutant CD38 molecule lacking the cytoplasmic domain, demonstrating that the latter is not required for CD38-mediated signaling in immature lymphoid cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IL-3-dependent murine pro-B cell line Ba/F3 19 and the human B lymphoblastic leukemic cell lines RS4;11 20 and OP-1 21 were available in our institution. Ba/F3 was cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with IL-3 (25 U/ml; from CHO cells expressing the murine IL-3 gene), 10% FCS (BioWhittaker), L-glutamine, and antibiotics. For RS4;11 and OP-1 we used an identical tissue culture medium without IL-3. To prepare bone marrow-derived stromal layers, we collected mononucleated cells from donor-derived normal marrow. The cells were separated on a density gradient (Lymphoprep, Nyegaard, Oslo, Norway) and washed three times in RPMI 1640. Stromal layers were prepared in flat-bottom 96-well plates (Costar, Cambridge, MA) and fed with RPMI 1640, 10% FCS, and 10-6 M hydrocortisone (Sigma, St. Louis, MO), as previously described 14, 22, 23, 24, 25, 26 .
Anti-CD38 mAbs comprised T16 (IgG1; Immunotech, Westbrook, ME), THB7 (IgG1; American Type Culture Collection, Manassas, VA), and IB4 (IgG2a) and IB6 (IgG2b; gifts from Dr. F. Malavasi, University of Ancona, Ancona, Italy). F(ab')2 of IB4 (from Dr. Malavasi), and Fab and F(ab')2 of THB7 (prepared in our laboratory) were used in some experiments. The purity of the latter reagents was verified by SDS-PAGE and was corroborated by the lack of staining seen in CD38+ RS4;11 cells when a goat anti-mouse Ig Fc antiserum was used as a secondary Ab. Rat anti-mouse LFA-1 (I21/7.7; gift from Dr. M. Blackman, St. Jude Children’s Research Hospital, Memphis, TN), rat anti-mouse CD71 (C2F2; PharMingen, San Diego, CA), and nonreactive mouse control Ig (Becton Dickinson, San Jose, CA) were also used. mAb to phosphotyrosine (4G10) was obtained from Upstate Biotechnology (Lake Placid, NY). Polyclonal anti-PI 3-K p85 was purchased from Transduction Laboratories (Lexington, KY). Polyclonal goat antiserum to TEC and rabbit antiserum to CBL were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antiserum to TEC was previously described 27 .
DNA constructs and electroporation conditions
The CD38 expression vector was constructed by excising the human
CD38 cDNA fragment from pCDM:CD38 (a gift from Dr. D. G. Jackson,
Oxford, U.K.) with XbaI and inserting it into an
XbaI-cleaved pEF-BOS mammalian expression vector (a gift
from Dr. S. Nagata, Osaka, Japan). Mutant CD38 with the cytoplasmic
tail deleted (CD38 
1–20) was constructed by subcloning the
XbaI fragment of human CD38 cDNA into pBluescript II
SK+ (Stratagene, La Jolla, CA). The pBluescript-CD38
contained a unique Eco47III site at nucleotide 241 of the
CD38 cDNA and a SpeI site in the multiple cloning site of
the vector. Thus, a SpeI-Eco47III fragment was
generated by PCR amplification (25 rounds) using 5'-GGACTAGTAT
GAGAGCCCAACTCTGTCTTGG-3' as a 5' primer, 5'-TCTCGGGAA AGCGCTTGGTGGT-3'
as a 3' primer, and pBluescript-CD38 as a template. The PCR-generated
fragment began with an SpeI site, which was followed by the
CD38-coding sequence including ATG as an initiation codon, three
nucleotides encoding for one amino acid (Arg) of the cytoplasmic
domain, and the entire transmembrane and extracellular domains through
the Eco47III site. The fragment was digested with
SpeI and Eco47III, and then used to replace the
SpeI-Eco47III portion of pBluescript-CD38. The
resulting plasmid was digested with SpeI and
XbaI, and the resulting cDNA fragment was inserted into
pEF-BOS. The introduced mutation was confirmed by DNA sequence
analysis.
Each expression plasmid (32 µg) was electroporated into 5 x 106 Ba/F3 cells with 3.2 µg of a second plasmid (pSTneoB) using a gene pulsar apparatus (Bio-Rad, Richmond, CA) set at 960 µF and 290 V. Cells were cultured for 24 h in RPMI 1640 plus additives (see above). Transfected cells were selected after 2 wk of culture in the presence of 1 mg/ml G418 (Life Technologies, Gaithersburg, MD). Individual clones were obtained by single-cell sorting using a FACSVantage flow cytometer equipped with an automatic cell deposition unit (Becton Dickinson). Individual clones were expanded and screened for the cell surface expression of human CD38 by labeling with anti-CD38 conjugated to FITC and analysis with a FACScan flow cytometer equipped with the CellQuest software (Becton Dickinson). Three clones for each transformant were selected and characterized in detail.
Immunoprecipitation, SDS-PAGE, and Western blotting
Immunoprecipitation was performed essentially as previously described 10, 11, 12, 13 . Briefly, after exposure to anti-CD38 Ab or control Ig (5–10 µg/ml), cells were lysed in 1 ml of ice-cold lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% (v/v) Triton X-100, 5 µg/ml aprotinin, 1 mM PMSF, 1 mM EDTA, and 1 mM Na3VO4) and centrifuged at 20,000 x g for 20 min at 4°C. Supernatants were precleared by 1 h of protein A-Sepharose treatment (20 µl of a 50% slurry). Abs were then added to the cleared lysates, which were incubated at 4°C for 1–2 h. The immune complexes were collected by using protein A-Sepharose.
For SDS-PAGE, cell lysates and immunoprecipitates were resuspended in sample buffer (10% (v/v) glycerol, 5% 2-ME, 3% (w/v) SDS, 65 mM Tris-HCl (pH 6.8), and 0.002% (w/v) bromophenol blue) and separated on a 7.5% acrylamide gel 10, 11, 12, 13 . After transfer, nitrocellulose filters were incubated first in 5% nonfat dry milk in 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% Tween-20 for 2 h and then with primary Abs for 1 h. After washing in 20 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% Tween-20, the filters were incubated for 1 h with horseradish peroxidase-conjugated sheep anti-mouse Ig or donkey anti-rabbit Ig (Amersham, Arlington Heights, IL). The filters were then washed, incubated with enhanced chemiluminescence detection reagents (Amersham), and exposed to Kodak BioMax MR film (Eastman Kodak, Rochester, NY). For reprobing, the filters were stripped and then reblocked, washed, and reprobed. All experiments were repeated at least three times.
Cell culture studies
Before each experiment, we removed the media from cultured
stromal cells and washed the adherent cells with RPMI 1640 to remove
hydrocortisone fully. Ba/F3 cells transfected with cDNA encoding the
entire CD38 molecule, with cDNA encoding the mutant lacking the
cytoplasmic tail (CD38 1–20), or with the vector only were
resuspended in fresh tissue culture medium with IL-3 (see above). Two
hundred microliters of each cell suspension (5 x 104
cells/ml) were then seeded onto marrow stromal cells. In parallel
experiments, transfected Ba/F3 cells were placed in the empty wells of
a 96-well flat-bottom microtiter plate. For culture experiments,
anti-CD38 Abs and nonreactive control Ig were dialyzed in PBS,
sterile-filtered, and used at 2–10 µg/ml. ß-NAD and hyaluronic
acid (from human umbilical cord) were purchased from Sigma and used at
the concentrations indicated in Results. All cell cultures
were incubated at 37°C in 5% CO2 with 90% humidity. At
the termination of cultures, plates were observed with an inverted
microscope. In some experiments cells were pretreated with the tyrosine
kinase inhibitor herbimycin A, the PI 3-K inhibitor wortmannin, or
their diluent DMSO (all from Sigma) for 1 h before addition of
Abs, at the concentrations indicated in Results.
Cell counting and assessment of cell death
After culture, cells were harvested by vigorous pipetting, suspended in PBS, and passed through a 19-gauge needle to disrupt clumps. Viable cells in culture were enumerated by flow cytometry, as previously described 14, 22, 23, 24, 25, 26 . To detect phosphatidylserine residues exposed on the cell surface (a marker of apoptosis) 28 , we labeled cells with FITC-conjugated annexin V (Trevigen, Gaithersburg, MD), following the manufacturer’s instructions. In these experiments, cell membrane permeabilization was revealed by labeling cells with 5 µg/ml of propidium iodide (Trevigen) for 15 min at 20°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clonal Ba/F3 cells expressing high levels of human CD38 were
obtained after transfection and single cell sorting. In the selected
clones, levels of cell surface CD38 expression were similar or higher
than those measured on human immature B cells (Fig. 1
). By contrast, mock-transfected Ba/F3
cells did not react with anti-human CD38 Abs.
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). Tyrosine phosphorylation was
also induced when T16 was replaced with another anti-CD38 Ab, THB7
(not shown). Time-course experiments showed that CD38-induced tyrosine
phosphorylation was rapid and transient (Fig. 3). Tyrosine phosphorylation was detected
as early as 1 min after exposure of cells to the anti-CD38 Ab and
gradually decreased thereafter. This kinetic profile of tyrosine
phosphorylation was similar to that induced by CD38 ligation in human
immature B cells 11 . Moreover, the pattern of tyrosine-phosphorylated
bands was remarkably similar (Fig. 4).
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) and CBL (not shown) proteins was
observed after CD38 ligation, while no substantial changes in the
levels of PI 3-K p85 tyrosine phosphorylation were observed. In these
experiments, however, several tyrosine-phosphorylated proteins
coprecipitated with PI 3-K p85 in cells exposed to anti-CD38 (Fig. 6), suggesting that CD38 ligation induces
association of p85 with other intracellular proteins.
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Ligation of human CD38 with T16 (IgG1), THB7 (IgG1), IB4 (IgG2a),
and IB6 (IgG2b) induced marked homotypic aggregation in cultures of
Ba/F3 cells. Aggregation became distinguishable after 2 h of
exposure to the Ab and was maximal after 24 h (Fig. 7). To determine whether Fc receptor
engagement was required for CD38-mediated cell aggregation, we
performed experiments with anti-CD38 reagents lacking the Fc
portion. F(ab')2 of THB7 and IB4 induced aggregation
identical with that seen with whole Abs (Fig. 7), thus excluding
Fc-mediated signaling. Notably, Fab of THB7 also induced cell
aggregation, suggesting that receptor cross-linking was not required.
By contrast, anti-LFA-1, anti-CD71 (both reactive with Ba/F3
cells), and control Ig did not cause cell aggregation.
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Ligation of CD38 in stroma-supported cultures of human immature lymphoid cells inhibits cell proliferation and induces cell death 14 . To determine whether a similar effect would occur in the transfectants, we cultured these cells on bone marrow-derived stroma in the presence of anti-CD38 Ab (T16). In seven experiments, the mean cell recovery after 3 days of culture in the presence of anti-CD38 Ab (T16) was 10.5 ± 9.2% (±SD) of the mean cell recovery in parallel cultures containing an isotype-matched nonreactive Ab. Cell recovery was also suppressed by another anti-CD38 Ab (THB7), although less markedly (37.5 ± 7.6% of control cultures in three experiments). The inhibitory effects of CD38 ligation on cell recovery could also be observed when anti-CD38 Ab (THB7) was used in an F(ab')2 form. In three experiments, cell recovery was 42.5 ± 6.2% of the recovery in parallel control cultures, an inhibitory effect similar to that observed with the whole THB7 molecule.
Decreased cell recovery in cultures containing anti-CD38 was at
least in part caused by cell death. After 24 h of CD38 ligation
there was a reduction in cell size accompanied by exposure of
phosphatidylserine residues on the cell membrane followed by surface
membrane permeabilization, compatible with apoptosis (Fig. 8).
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Relationship between biochemical and cellular effects triggered by CD38 ligation
To determine whether the homotypic aggregation observed after CD38
ligation was dependent on the increased tyrosine kinase activity, we
added anti-CD38 to cultures of CD38-transfected Ba/F3 cells exposed
to herbimycin A, a tyrosine kinase inhibitor. At 20 µM, herbimycin A
completely abrogated cell aggregation (Fig. 9). In parallel experiments, herbimycin A
was replaced with wortmannin (100–500 nM), a PI 3-K inhibitor. In
these cultures, CD38-mediated aggregation was delayed; definitive cell
aggregates begun to appear after 5 h or more of exposure to CD38,
but after only 2 h in cultures without wortmannin (Fig. 9).
However, after 24 h, cultures with and without wortmannin were
indistinguishable. These results indicate that cell aggregation depends
on the induction of tyrosine phosphorylation and suggest the
involvement of PI 3-K in this signaling pathway.
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The cytoplasmic portion of CD38 is not required for signaling in lymphoid cells
Human CD38 has a short cytoplasmic domain that has no tyrosine residues and no known motifs 1 . To test whether this domain is required for the cellular and signaling events triggered by CD38 ligation in lymphoid cells, we prepared a construct encoding a truncated form of CD38 lacking all but the first amino acid proximal to the transmembrane portion, and expressed this construct in Ba/F3 cells.
In cells expressing the mutant CD38 (Fig. 1
), ligation of CD38 induced
tyrosine phosphorylation with intensity and kinetics similar to those
seen in cells expressing the entire protein (Fig. 10). Consistent with these findings,
ligation of CD38 in these cells induced vigorous cell aggregation that
was maximal after 24 h of incubation with the Ab (not shown).
Moreover, cell growth was inhibited when Ba/F3 cells expressing the
CD38 cytoplasmic mutant were exposed to anti-CD38 Ab. In two
separate experiments, cell recoveries after 3 days of culture on stroma
in the presence of anti-CD38 (T16) were 5.6 and 15.6% of cell
recovery in parallel cultures containing an isotype-matched control Ab.
Taken together, these results indicate that the cytoplasmic domain of
CD38 is not essential for signal transduction in immature lymphoid
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise changes induced by Ab ligation in the expression, lateral association, or conformation of the CD38 molecule that result in signal transduction are still unclear. It is unlikely that changes in the CD38 enzymatic activity participate in this effect, as NAD hydrolysis and production of cyclic ADPR are not affected by binding of anti-CD38 to the surface of immature B cells 14 . One possibility is that Ab binding induces CD38 internalization, which, in turn, could trigger signaling pathways, perhaps via protein ribosylation 31 or mobilization of Ca2+ from intracellular stores 9, 32 . Indeed, capping and internalization of CD38 following exposure to anti-CD38 Abs have been documented 33 , although in immature B cells this response is clearly less prominent than that of other transmembrane molecules, such as CD19, under similar circumstances 10 .
The hypothetical structure of CD38, as predicted from the crystal structure of the homologous molecule ADPR cyclase, comprises a dimer capable of hinge motion 15 . Prasad et al. postulated that either enzymatic activity or ligand binding could cause conformational changes in the dimer. In turn, these could juxtapose tyrosine kinases bound to the cytoplasmic portion of CD38, triggering the tyrosine kinase cascade. We found that CD38-mediated signaling in Ba/F3 did not require the cytoplasmic portion of CD38, a result that agrees with the observations of Inoue et al. in myeloid cells 34 . Taken together, these data suggest a more complex model, in which an as yet unknown transmembrane molecule (lineage specific or common to all hemopoietic lineages) is linked to the extracellular domain of CD38 and transduces conformational changes in CD38. However, we have not been successful to date in identifying tyrosine-phosphorylated proteins associated with CD38 in Ba/F3 cells.
The biochemical events triggered by CD38 ligation in Ba/F3 cells are accompanied by vigorous homotypic aggregation followed by cell death. Notably, aggregation was suppressed by inhibitors of tyrosine kinase and PI 3-K, implying a cause-effect relationship between biochemical and cellular events mediated by CD38. It is still not known whether aggregation was caused by de novo expression of surface adhesion molecules or by other mechanisms, but it appeared to be independent of Ca2+ and Mg2+ flux, and did not involve LFA-1/ICAM-1 interaction. Homotypic aggregation could also be triggered by F(ab')2 and Fab of the CD38 Ab, indicating that is not dependent on Fc receptor cross-linking. In contrast, signaling triggered by anti-CD38 Abs in myeloid leukemic cells requires simultaneous binding of anti-CD38 Abs to Fc receptors 34 .
In summary, CD38 is a surface receptor that can mediate biochemical and cellular signals in human and murine immature B cells independently from its cytoplasmic domain. The results of this and other studies 14, 16, 35, 36, 37 imply the existence of natural ligands for CD38 that can induce events similar to those triggered by ligation of CD38 with mAbs. Hyaluronic acid and CD31 have been shown to bind CD38 and may well represent natural CD38 counter-receptors 29, 30 . Likewise, ß-NAD hydrolysis could provoke hinge motion in the CD38 dimer resulting in signal transduction. However, we failed to detect signals in Ba/F3 cells expressing human CD38 after exposure to ß-NAD, hyaluronate, or CD31. Thus, these molecules cannot account for the full range of cellular and biochemical effects mediated by CD38, suggesting the existence of other molecules capable of triggering CD38 signaling. In this respect, it is relevant that reducing agents can cause self-aggregation and internalization of the CD38 molecule 38 , suggesting alternative means of triggering CD38 signaling that warrant further exploration.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. D. Campana, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. E-mail address:
3 Abbreviations used in this paper: NAD, nicotinamide adenine dinucleotide; ADPR, adenosine diphosphate ribose; PI 3-K, phosphatidylinositol 3-kinase.
Received for publication July 27, 1998. Accepted for publication November 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and phosphatidylinositol 3-kinase. J. Immunol. 156:100.[Abstract]
/-associated protein-70 signaling pathways in Jurkat T lymphocytes. J. Immunol. 159:193.[Abstract]
II receptors in human myeloid cells. J. Immunol. 159:5226.[Abstract]
, IL-6, granulocyte-macrophage colony-stimulating factor and IL-10 cytokines after activation of human purified T lymphocytes upon CD38 ligation. Cell. Immunol. 173:192.[Medline]
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