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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1324-1333
Mice Deficient for the Ecto-Nicotinamide Adenine Dinucleotide
Glycohydrolase CD38 Exhibit Altered Humoral Immune Responses
By
Debra A. Cockayne,
Tony Muchamuel,
J. Christopher Grimaldi,
Hélène Muller-Steffner,
Troy D. Randall,
Frances E. Lund,
Richard Murray,
Francis Schuber, and
Maureen C. Howard
From the Department of Immunology, DNAX Research Institute of
Molecular and Cellular Biology, Palo Alto, CA and the Laboratoire de
Chimie Bioorganique, Faculté de Pharmacie, Université Louis
Pasteur, Strasbourg-Illkirch, France.
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ABSTRACT |
CD38 is a membrane-associated ecto-nicotinamide adenine dinucleotide
(NAD+) glycohydrolase that is expressed on multiple
hematopoietic cells. The extracellular domain of CD38 can mediate the
catalysis of NAD+ to cyclic adenosine
diphosphoribose (cADPR), a Ca2+-mobilizing second
messenger, adenosine diphosphoribose (ADPR), and nicotinamide. In
addition to its enzymatic properties, murine CD38 has been shown to act
as a B-cell coreceptor capable of modulating signals through the B-cell
antigen receptor. To investigate the in vivo physiological function(s)
of this novel class of ectoenzyme we generated mice carrying a null
mutation in the CD38 gene. CD38 / mice showed a
complete loss of tissue-associated NAD+ glycohydrolase
activity, showing that the classical NAD+ glycohydrolases
and CD38 are likely identical. Although murine CD38 is expressed on
hematopoietic stem cells as well as on committed progenitors, we show
that CD38 is not required for hematopoiesis or lymphopoiesis. However,
CD38/ mice did exhibit marked deficiencies in
antibody responses to T-cell-dependent protein antigens and augmented
antibody responses to at least one T-cell-independent type 2 polysaccharide antigen. These data suggest that CD38 may play an
important role in vivo in regulating humoral immune responses.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE TYPE II membrane-associated
glycoprotein CD38 is the prototypic member of the class of adenosine
diphosphoribose (ADPR) transfer enzymes known as nicotinamide adenine
dinucleotide (NAD+) glycohydrolases.1
CD382-4 and the classical NAD+
glycohydrolases5,6 are multifunctional ectoenzymes able to
catalyze the net hydrolysis of NAD+ to nicotinamide and
ADPR,7 as well as to catalyze both the synthesis and
hydrolysis of cylic adenosine diphosphoribose (cADPR). Thus, in
addition to their intrinsic NAD+ hydrolysis (NADase)
activity, these enzymes possess both adenine diphosphate (ADP)-ribosyl
cyclase and cADPR hydrolase activities. The possibility that these
ectoenzymes might be involved in the metabolism of cADPR is intriguing
given that cADPR is a potent inositol triphosphate (IP3)-independent
Ca2+-mobilizing agent8-10 believed to be
involved in Ca2+-induced Ca2+ release in
multiple mammalian cell types, including pancreatic  cells11 and neuronal cells.12,13 cADPR has also
been shown to augment the proliferative response of activated murine B
lymphocytes,2 as well as to mediate the Ca2+
release associated with adenosine triphosphate (ATP)-activated potassium currents in alveolar macrophages,14 suggesting
that cADPR may function as a signaling second messenger in multiple hematopoietic cell types.
All of the mammalian NAD+ glycohydrolases that have been
identified to date, including CD38,15 the bone marrow
stromal cell surface molecule BP-3/BST-1,16,17 and the
T-cell differentiation marker RT-6,18 are highly expressed
on cells of hematopoietic origin. Although originally identified as a
lymphocyte surface antigen,19 CD38 is now known to be
expressed by many hematopoietic cell types including murine bone
marrow-derived stem cells, B cells throughout ontogeny, subsets of
developing and mature T cells, natural killer cells, macrophages,
granulocytes, neutrophils, and platelets, as well as by pancreatic cells and in organs such as the brain, liver, and
kidney.15,20-22 Clinically, CD38 has been recognized for
many years as a marker for certain human malignancies,19,23,24 and recent studies have suggested
that CD38 may be a useful prognostic indicator of human
immunodeficiency virus disease progression.25,26
In addition to serving as a marker for different cell types, CD38 has
been shown to have a number of immunologically relevant functional
activities in vitro. For example, CD38 has been implicated as an
adhesion molecule capable of binding endothelial cells.20 In addition, ligation of CD38 by specific monoclonal antibodies can
induce cytokine production by human peripheral blood mononuclear cells,27 enhance murine macrophage antigen presenting cell
(APC) function (N. Solvason et al, manuscript in
preparation), inhibit B lymphopoiesis,28 protect mature B
lymphocytes from apoptosis,20,29 and induce proliferation
of mature T and B lymphocytes.15,20 More recently, murine
CD38 was shown to act as a coreceptor for the B-cell antigen receptor
(BCR) capable of either augmenting or depressing BCR-mediated B-cell
activation30,31 in a manner similar to that observed for
CD19.32 CD38, in combination with interleukin-5 (IL-5), has
also been shown to be a potent costimulatory signal for IgM and IgG1
production by mature splenic B lymphocytes,33 further
implicating CD38 in the regulation of signals delivered through the
BCR. Although the exact mechanism(s) by which CD38 modulates lymphocyte
responses is still to be determined, it has been shown that signaling
through CD38 in various lymphoid and myeloid cell populations involves
the activation and tyrosine phosphorylation of numerous intracellular
substrates including phospholipase C- , c-cbl, syk, btk, lyn, fyn,
and ZAP-70.33-35 Interestingly, CD38 ligation on human
immature B cells was recently shown to induce tyrosine phosphorylation
of CD19 and the subsequent association of CD19 with lyn and
phosphatidylinositol 3 (PI3)-kinase.36 Taken together,
these data suggest that CD38 might use the signal transduction pathways
associated with CD19 to mediate certain B-cell responses. Even more
recent data have shown that CD38 can potentiate signals through
(Gi)-coupled chemotactic formyl-Met-Leu-Phe receptors on human
myeloid cells37 suggesting that G-protein-coupled signal
transduction pathways might also be involved in CD38-mediated intracellular signaling.
In the present study we generated CD38-deficient mice to elucidate the
biological function(s) of CD38 in vivo, as well as to extend our
understanding of the functional heterogeneity and physiological role of
NAD+ glycohydrolases. Our initial studies presented here
show that CD38/ mice exhibit altered humoral
immune responses, supporting our recent in vitro findings that CD38 is
a coreceptor for the BCR.30
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MATERIALS AND METHODS |
Generation of CD38 gene-targeted mice.
The murine CD38 gene from exons 1 through 6 was cloned from a 129SvEv
mouse  genomic library (Stratagene, La Jolla, CA). The CD38
targeting vector was generated by subcloning isolated genomic fragments
into a vector containing the neomycin resistance gene, and the HSV-TK
gene. 129Ola-derived E14-1 embryonic stem (ES) cells (kindly provided
by Drs Klaus Rajewsky and Ralf Kuhn, Institute for Genetics, University
of Cologne, Cologne, Germany) were transfected with a
Cla I linearized targeting vector, and 2 of 300 (1 of 150)
G418r/gancr colonies (310 mg/mL active G418, 2 mmol/L gancyclovir) screened were determined to be correctly targeted
by Southern blot analysis. CD38+/ ES clones were
injected into C57BL/6J blastocysts, blastocysts were implanted into
pseudopregnant B6.CBA F1 females, and resulting chimeras were crossed
to C57BL/6J mice to establish germline transmission. CD38+/ F1 mice were intercrossed, and F2 breeding
pairs were established from CD38+/+ and
CD38/ mice. 129Ola × C57BL/6J F3 mice
were used for all experiments shown. Both homologous recombinant clones
(236 and 2832) transmitted the CD38 mutation to the germline, and
experiments confirmed in both clones are indicated.
Assay of NAD+ glycohydrolase activity.
Membrane fractions were prepared from spleen, liver, and brain by
homogenization of whole organs at 4°C in 1 mL of 50 mmol/L potassium phosphate buffer, pH 6.8, using a glass Dounce
homogenizer. The total protein concentration was determined by the
BCA* protein assay (Pierce, Rockford, IL) by using bovine
serum albumin as the standard. NAD+ glycohydrolase and
nucleotide pyrophosphatase activity determinations were made using
sensitive fluorometric enzyme assays. The reactions were performed at
37°C, in a 2-mL final volume of 50 mmol/L potassium phosphate
buffer, pH 6.8, and the reaction progress was followed by monitoring
the increase in fluorescence at 410 nm (excitation at 310 nm). This pH
value minimizes the self-inactivation of the murine NAD+
glycohydrolase.1 NAD+ glycohydrolase activity
was measured with 1, N6-etheno-NAD+ as
substrate under saturating conditions (250 µmol/L final
concentration).38 Nucleotide pyrophosphatase activity was
measured under the same experimental conditions using as substrate
pyridine 1, N6-ethenoadenine dinucleotide (62.5 µmol/L
final concentration), a poorly NADase-hydrolyzable
analog.39,40 This activity was determined because it can
contribute to the hydrolytic cleavage of 1, N6-etheno-NAD+, potentially leading to an
overestimation of NADase activity. The NAD+ glycohydrolase
values shown in Table 1 represent real
NAD+ glycohydrolase activities, derived by subtracting the
nucleotide pyrophosphatase activity from the total measured
NAD+ glycohydrolase activity. Enzyme activity measurements
determined by fluorometric enzyme assays were confirmed by analysis of
the reaction products by high-performance liquid chromatography (HPLC). HPLC analysis was performed on Microcon 10 (Amicon, Beverly,
MA) concentrated aliquots of the reaction media by using a
Waters HPLC system (Waters Chromatography, Milford, MA). Chromatography was performed on a 300 × 3.9 mm µBondpack C18
column (Waters Chromatography) operated at ambient temperature at a
flow rate of 1 mL/min. The compounds were eluted isocratically with a
10-mmol/L ammonium phosphate buffer, pH 5.5, containing 1.5% (vol/vol)
acetonitrile and were detected by ultraviolet absorbance at 260 nm.
Long-term reconstitution assays.
The repopulating ability of CD38/ bone marrow
was determined in a competitive repopulation assay.41 Bone
marrow was obtained from the femurs and tibias of CD38+/+
and CD38/ (Ly5.2) 129Ola x C57BL/6J mice.
C57BL/6J (Ly5.1) recipient mice were irradiated with 950 rad from an
x-ray source operated at 200 kV delivering 85 rad/min. Mice were
irradiated in a split dose administered 4 to 5 hours apart. A total of
5 × 106 bone marrow cells from each source were
injected retroorbitally into irradiated recipients. After irradiation
and reconstitution, the recipient animals were maintained on
antibiotic-containing water (polymixin B sulfate 106 U/L
and neomycin sulfate 1.1 g/L) for at least 6 weeks. Reconstituted mice
were analyzed at 12 weeks by fluorescence-activated cell sorting (FACS)
staining of peripheral blood for the lineage antigens B220, CD3, Mac-1,
and GR-1, as well as for the donor marker Ly5.2. The percentage of
Ly5.2+ cells in each lineage was determined by integration
by using FACS Desk software (Stanford University, Stanford,
CA).
Immunizations and measurement of antigen-specific serum
immunoglobulin levels.
For immunization with T-cell-dependent (TD) protein antigens, 8- to
10-week-old mice were immunized intraperitoneally with either 10 µg
or 1 µg of alum-precipitated 2,4,6-trinitrophenyl conjugated to
keyhole limpet hemocyanin (TNP5-KLH), or 10 µg of complete Freud's adjuvant (CFA)-precipitated
4-hydroxy-3-nitrophenylacetyl coupled to chicken -globulin
(NP20-CG) on day 0 for a primary immunization, and again on
day 21 (TNP5-KLH) or day 28 (NP20-CG) for a
secondary immunization. Mice were bled, and serum was collected on days
0, 7, 10, 14, 21, 28, 31, and 35 for the TNP5-KLH
immunizations, and on days 0, 7, 10, 14, 28, 38, and 42 for the
NP20-CG immunizations. TNP-specific IgM, IgG1, IgE, IgG2a,
IgG2b, IgG3, and IgA antibody titers were assayed by enzyme-linked
immunosorbent assay (ELISA)42 (limit of detection 10 ng/mL). NP-specific -bearing and IgG1 antibody titers were assayed
by ELISA43 (limit of detection 64 ng/mL). For immunization
with T-cell-independent type 2 (TI-2) polysaccharide antigens, 8- to
10-week-old mice were immunized intraperitoneally with 50 µg of
 1 3 dextran or 10 µg of 4-hydroxy-3-nitrophenylacetyl conjugated to Ficoll (NP27-Ficoll). Mice were bled, and
serum was collected on days 0 and 7. The titers of -bearing
13 dextran-specific serum antibody were determined by
ELISA,44 with the modification that the secondary goat
antimouse antibody was conjugated to horseradish peroxidase (1:1,000;
Southern Biotechnology Associates Inc, Birmingham, AL). The titers of
-bearing NP-specific serum antibody were determined by
ELISA.43
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RESULTS |
Establishment of CD38-deficient mice and analysis of
NAD+ glycohydrolase activity.
Homologous recombination in ES cells resulted in two ES clones in which
exons encoding the putative active site for CD38 NAD+
hydrolysis activity45 were replaced by the neomycin
resistance gene (Fig 1A and B). Resultant
CD38/ mice lacked CD38-specific mRNA
transcripts (data not shown) and immunoreactive CD38 (Fig 1C). In
addition, B cells from these mice were unresponsive to the
proliferative effects of anti-CD38 antibodies (data not shown).
CD38/ mice were comparable with
CD38+/+ littermate controls in appearance and behavior and
were capable of living to at least 8 months of age without the
development of overt histological or pathological abnormalities.
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| Fig 1.
Generation of CD38 gene-targeted mice. (A) Schematic
diagram of the CD38 genomic locus, targeting vector, and targeted
locus. Exons, lengths of regions of CD38 homology, lengths of
diagnostic restriction fragments, and the position of the exon
4-specific probe are indicated. Restriction enzyme sites are indicated
(H, HindIII; C, Cla I; S, Sac I; S*,
Sac I polymorphism between the 129SvEv and BALB/c strains of
mice). Arrows indicate the transcriptional orientation of the CD38,
HSV-TK, and neomycin resistance genes. Homologous recombination
resulted in replacement of a 1.6-kb CD38 genomic region that included
the putative NAD+ glycohydrolase enzyme catalytic site.
(B) Southern blot analysis used for screening genomic DNA from
recombinant clones (not shown), and 129Ola × C57BL/6J
CD38+/+, CD38+/, and
CD38/ mice. Probing of blots with a 174-bp polymerase
chain reaction-derived exon-4-specific probe (top panel) shows the
predicted 2-kb and 10-kb Sac I fragments diagnostic of the CD38
endogenous and mutant alleles, respectively. As a consequence of the
Sac I polymorphism in the BALB/c locus, a Sac I digest
of BALB/c genomic DNA provides a control for the approximate size of
the mutant allele fragment. Probing of blots with a neo probe (lower
panel) shows hybridization specifically to the 10-kb Sac I
fragment characteristic of the mutant allele. (C) FACS staining of
total splenocytes with the phycoerythrin (PE)-conjugated rat antimouse
CD38 monoclonal antibody NIMR5 (0.5 µg/mL, shaded), or an appropriate
PE-conjugated rat IgG2a isotype control (unshaded), was performed
according to standard methods.
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In an attempt to understand the relationship between the classical
NAD+ glycohydrolase activities that have been extensively
studied1 and the more recently isolated NAD+
glycohydrolases, such as CD38,2,3
BP-3/BST-1,16,17 and RT6,46-48 we assessed the
status of NAD+ glycohydrolase activity in
CD38/ mice. NAD+ hydrolysis
activities in the spleen, liver, and brain, organs known to be rich in
classical NAD+ glycohydrolase activity, were determined by
using a fluorometric assay with 1, N6-etheno-NAD+ as substrate. The results in
Table 1 show that in the spleens of CD38/
mice the NAD+ glycohydrolase activity was dramatically
reduced to less than 1% of the activity detected in the spleens of
CD38+/+ littermate controls. The remaining NAD+
hydrolysis activity present in the spleens of
CD38/ mice presumably reflects a minor
contribution from other NAD+ glycohydrolases previously
detected in this tissue.16-18 However, it is unlikely that
the residual activity in the spleens of CD38/
mice is contributed by B lymphocytes since we found no detectable NAD+ glycohydrolase activity remaining in sort
purified B220+ splenic B cells from these mice
(data not shown). In contrast to the spleen, in the liver and brain of
CD38/ mice no NAD+ glycohydrolase
activity was detected. The total lack of NAD+
glycohydrolase activity in the liver and brain of these mice is
consistent with the fact that CD38 is the only NAD+
glycohydrolase that has been detected in these tissues to
date.16,18 Taken together, these data provide the first
convincing evidence that, at least in the spleen, liver, and brain,
CD38 and the classical NAD+ glycohydrolases are likely to
be identical, and identify CD38 as the predominant NAD+
glycohydrolase in these tissues.
Effect of CD38 deficiency on hematopoiesis in vivo.
Because CD38/ mice are dramatically depleted
of NAD+ glycohydrolase activity, they provide a valuable
model for elucidating the physiological role of this class of enzyme,
particularly within the hematopoietic compartment where CD38 is widely
expressed.15,22 Studies in both the human49-51
and murine22 systems have shown that CD38 is a useful
marker for identifying populations of hematopoietic progenitor cells.
Furthermore, in the murine system, CD38 can be used in combination with
other markers to define a population of bone marrow stem cells that
possess long-term reconstituting potential. These observations raise
the interesting possibility that, in addition to serving as a useful
marker for bone marrow progenitor cells, CD38 might play a role in
hematopoietic stem cell function. To address this question, competitive
repopulation assays were performed whereby irradiated
Ly5.1+ recipient mice were competitively repopulated with
equal numbers of bone marrow cells derived from Ly5.1+
congenic mice, and either Ly5.2+ CD38+/+ or
Ly5.2+ CD38/ mice. The results in
Table 2 show that at 12 weeks
postreconstitution, bone marrow cells derived from
CD38/ mice had repopulated the peripheral
T-cell, B-cell, granulocyte, and macrophage lineages in a manner that
was indistinguishable from that observed with bone marrow cells derived
from CD38+/+ littermate controls.
Consistent with these findings, CD38/ mice
exhibited normal peripheral blood cell counts and hematopoietic organ
cellularities (data not shown). In addition, flow cytometric analysis
of tissues from 4- and 8-week-old CD38/ mice
indicated a normal distribution and ratio of immature
B220+IgM and mature
B220+IgM+ B cells in the bone marrow and
spleen, as well as B220+CD5+ B-1 B cells in the
peritoneal cavity (data not shown). T lymphocyte subpopulations
including CD4+CD8+ double positive thymocytes,
and CD4+ or CD8+ single positive thymocytes and
peripheral T cells were also present in normal numbers and ratios, as
were cells of the independent lineage of T-cell receptor
(TCR)+CD4CD8
thymocytes which uniquely express CD3852 (data not shown). Normal numbers of macrophages, granulocytes, and neutrophils were also
present in the bone marrow, spleen, and peritoneal cavity of
CD38/ mice (data not shown). Taken together,
these data indicate that despite the prevalence of enzymatically active
CD38 on a wide range of hematopoietic cells, this ecto-NAD+
glycohydrolase does not appear to be required for the development or
maintenance of either hematopoietic stem cells or committed lineage-specific cell populations.
In vivo modulation of TD and TI-2 antibody responses in
CD38-deficient mice.
Because CD38 has been implicated as a B lymphocyte coreceptor capable
of influencing both the threshold and the level of signaling through
the BCR,30 we assessed the immune response of
CD38/ mice after immunization with either
major histocompatibility complex (MHC) class II-restricted TD protein
antigens, or TI-2 polysaccharide antigens. In response to immunization
with 10 µg of the TD antigen TNP5-KLH in alum adjuvant,
CD38/ mice mounted normal hapten-specific
IgG2a, IgG2b, IgG3, and IgA responses, but decreased hapten-specific
IgM, IgG1, and IgE responses (Fig 2A and
data not shown). The most striking defect was in the ability of
CD38/ mice to mount or sustain a secondary
hapten-specific IgG1 response, with IgG1 titers consistently remaining
at the level observed after a primary immunization (Fig 2A, right
panel). Immunization with the same dose of a second TD antigen,
NP20-CG in alum adjuvant, produced similar results (Fig
2B). In addition, the impaired hapten-specific IgG1 response to both
TNP5-KLH and NP20-CG were confirmed in
CD38/ mice derived from a second,
independently targeted ES clone (data not shown).
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| Fig 2.
Immune responses of 129Ola × C57BL/6J F3
CD38/ mice (open symbols) and
CD38+/+ littermate controls (closed symbols) to
TD antigens. Titers of hapten-specific IgG1 serum antibody at various
time points after a primary (left), and secondary (right) immunization
with 10 µg alum-precipitated TNP5-KLH (A), 10 µg
alum-precipitated NP20-CG (B), or 10 µg
CFA-precipitated TNP5-KLH (C). Arrows
indicate the time of the primary and secondary immunizations. The data
shown are representative of one of eight TNP5-KLH in alum
immunization experiments, one of two NP20-CG in alum
immunization experiments, and one of three TNP5-KLH in CFA
immunization experiments, in which each experiment included five to
eight mice per group.
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The impaired TD antibody responses in CD38/
mice could reflect impaired B-cell class switching, decreased B-cell
responsiveness, insufficient T-cell priming/activation, inefficient
antigen presentation, or some combination of these possibilities.
However, serum immunoglobulin isotype levels were normal in
CD38/ mice (data not shown), suggesting that
B lymphocytes from these mice are not generally impaired in their
ability to undergo immunoglobulin class switching. In addition, B cells
from CD38/ mice, when stimulated in vitro
with either lipopolysaccharide, anti-CD40 antibodies, or
anti-immunoglobulin antibodies, proliferated and upregulated expression
of MHC class II, CD80, and CD86 (data not shown). T cells from
CD38/ mice also proliferated in vitro in
response to either anti-CD3, or anti-CD3 plus anti-CD28 antibody
stimulation (data not shown). These data indicate that both B and T
lymphocytes from CD38/ mice respond normally
to in vitro polyclonal activation signals, as well as to
contact-dependent signals important for cognate T- or B-cell
interactions.
As an alternative consideration, the impaired antibody responses
observed in CD38-deficient mice may reflect an alteration in
sensitivity to BCR signaling, consistent with our data that CD38 serves
as a coreceptor for the BCR.30 To test this possibility, the same types of immunization experiments described previously were
performed by using TNP5-KLH in Freund's adjuvant, which is a much more potent adjuvant than alum.53 Under these
experimental conditions we found no differences in the primary or
secondary antibody responses of CD38/ mice
compared with CD38+/+ littermate controls (Fig 2C). In one
experiment in which mice were similarly immunized with
NP20-CG in Freund's adjuvant, we found that the primary
antibody responses of CD38/ mice were also
not significantly different than that observed with CD38+/+
littermate controls (data not shown). To pursue this further, we next
measured the response of CD38/ mice to
immunization with decreasing amounts of TNP5-KLH in alum. Interestingly, the hapten-specific antibody responses were even more
impaired after immunization with 1 µg of TNP5-KLH in alum compared with that observed after immunization with 10 µg of antigen. As shown in Fig 3, immunization with a
lower dose of antigen now revealed marked deficiencies in the ability
of CD38/ mice to mount primary
hapten-specific IgM, IgG1, and IgE responses. Consistent with the
results shown in Fig 2, these mice failed to mount significant
secondary hapten-specific antibody responses to either 1 µg or 10 µg of antigen (data not shown). Taken together, these results
indicate that, at least in response to the antigens tested, immune
responses in CD38/ mice are critically
dependent on the dose and the strength of the immunizing antigen.
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| Fig 3.
Immune responses of 129Ola × C57BL/6J F3
CD38/ mice (open symbols) and CD38+/+
littermate controls (closed symbols) to a limiting dose of the TD
antigen TNP5-KLH in alum. In two of the eight
TNP5-KLH in alum immunization experiments described in Fig
2, the hapten-specific responses to different doses of antigen were
compared (1 µg compared with 10 µg). The data shown are
representative of these experiments, and illustrate the titers of
hapten-specific IgM, IgG1, and IgE serum antibody at various time
points after a primary immunization with 1 µg alum-precipitated
TNP5-KLH (10 µg, data not shown). Arrow indicates the
time of primary immunization.
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Because the loss of CD38 resulted in impaired TD immune responses, we
next examined the role of CD38 in TI-2 responses. In contrast to their
reduced antibody responses to TD antigens,
CD38/ mice showed elevated antibody responses
to the TI-2 antigen 13 dextran. In fact, immunization of
CD38/ mice with 13 dextran
produced an augmented antigen-specific total response that was, on
average, fivefold greater than that observed in CD38+/+
littermate controls (Fig 4A). Although this
TI-2 response was significantly increased in
CD38/ mice, the response to a second TI-2
antigen, NP27-Ficoll was unaltered (Fig 4B). One possible
explanation for this difference could be that while the
NP27-Ficoll response is mediated by conventional B cells,
the 13 dextran response is mediated by B-1 B
cells.54,55 However, CD38/ mice
showed no difference in absolute numbers or surface marker phenotype of
B-1 B cells compared with CD38+/+ littermate controls (data
not shown). Moreover, not all B-1 B-cell responses were augmented in
these mice because CD38/ mice showed no
upregulation of circulating autoantibody levels to antigens such as
double-stranded DNA even at 6 months of age (data not
shown). Taken together, these data suggest that CD38 plays a complex
role in regulating humoral immune responses in vivo.
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| Fig 4.
Immune responses of 129Ola × C57BL/6J F3
CD38/ mice (open symbols) and CD38+/+
littermate controls (closed symbols) to TI-2 antigens. Titers of
-bearing 13 dextran-specific serum antibody (A), or
-bearing NP-specific serum antibody (B) in individual mice at day 7 after immunization with 50 µg of 13 dextran, or 10 µg
of NP27-Ficoll, respectively. The 13 dextran
data shown represent the composite results from four independent
immunization experiments and include 17 CD38/ mice
(mean = 1131.1 µg/mL) and 17 CD38+/+ littermate
controls (mean = 247.3 µg/mL). The NP27-Ficoll data shown represent the composite results from three independent
immunization experiments and include 15 CD38/ mice
(mean = 172.4 µg/mL), and 13 CD38+/+ littermate
controls (mean = 233.9 µg/mL).
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DISCUSSION |
In the present study we describe the generation and analysis of a mouse
knockout model for the ecto-NAD+ glycohydrolase CD38. Our
analysis of these CD38/ mice has led to two
principal observations. First, these mice have enabled us to determine
that CD38 and the classical NAD+ glycohydrolases that have
been studied for many years,1 but whose function has
remained largely unknown, are likely identical molecules. Secondly, we
show that one prominent phenotype associated with CD38 deficiency is a
marked alteration in humoral immune responses to both TD and TI-2
antigens. Although one might have predicted a more critical role for
this widely in vivo distributed, multifunctional NAD+
glycohydrolase, these findings are nonetheless consistent with the
recognized role of CD38 as an immunoregulatory molecule and support our
previous in vitro observation that CD38 can act as a coreceptor for the
BCR, in a manner similar to that observed for other B-cell coreceptors
such as CD19.32
Several observations would have predicted an in vivo role for CD38 in
bone marrow hematopoiesis and B lymphopoiesis. CD38 is widely expressed
on cells of hematopoietic origin, including hematopoietic stem cells,
and studies in both the human49-51 and murine22
systems have used CD38 as a phenotypic marker for different hematopoietic progenitor subsets. In addition, functional experiments using anti-CD38 antibodies have shown that signaling through CD38 can
inhibit human B lymphopoiesis and expansion.28 However, analysis of CD38/ mice has shown that this
ecto-NAD+ glycohydrolase is not required for the
development or maintenance of either hematopoietic stem cells or
committed lineage-specific cell populations. The competitive
repopulation experiments shown in Table 2 showed that bone marrow
derived from CD38/ mice exhibited normal
repopulating ability. Consistent with these findings,
CD38/ mice exhibited a normal distribution
and ratio of B- and T-lymphocyte subpopulations, as well as normal
numbers of granulocytes, macrophages, and neutrophils (data not shown).
The fact that the hematopoietic compartment has remained unaltered in
CD38-deficient mice suggests that CD38 is not critical to hematopoiesis
or lymphopoiesis in mice. However, it is equally possible that in
CD38/ mice additional CD38-like
NAD+ glycohydrolases, such as the bone marrow stromal cell
surface molecule BP-3/BST-1,16,17 might compensate for CD38
function in vivo. Given that it is still unclear whether the enzymatic properties of molecules such as CD38 or BP-3/BST-1 are required for
their in vivo functional activity, it is too early to predict whether
such a functional compensation may be important to the maintenance of a
normal hematopoietic compartment. The generation of mice carrying
targeted mutations in both the CD38 and BP-3/BST-1 genes should enable
us to address this issue.
In contrast, CD38 does appear to play a functional role in mature
lymphocytes. CD38/ mice exhibited marked
deficiencies in antibody responses to TD protein antigens, and
augmented antibody responses to at least one TI-2 polysaccharide
antigen. In response to two separate TD antigens,
CD38/ mice failed to mount normal
hapten-specific IgG1 responses, and to a lesser extent IgM and IgE
responses (Figs 2 and 3 and data not shown), although the serum
immunoglobulin levels in these mice were normal (data not shown). These
data suggest that in vivo signals delivered through CD38 are not
critical for the maintenance of normal serum immunoglobulin, but
instead are important for maintaining antigen-driven responses of
specific immunoglobulin isotypes. Interestingly, recent experiments
from Yasue et al33 showed that CD38-mediated signaling in B
cells results in the preferential induction of specific immunoglobulin
isotypes. In this study, it was shown that ligation of murine splenic B
cells with anti-CD38 antibodies and IL-5 induced a marked induction of
IgM and IgG1 isotypes but not IgA or IgG2a. Because a similar stimulation of B cells with anti-CD38 and interleukin-4 (IL-4) did not
induce significant IgG1 production, it was suggested that there might
be two independent pathways for polyclonal IgG1 production: one that is
dependent on IL-4 and a second that is dependent on CD38 and
IL-5.33 Based on these observations, it is possible that
the diminished hapten-specific IgM and IgG1 responses observed in
CD38/ mice may be caused by the loss of a
CD38-dependent IL-5 signaling pathway.
The deficiencies in hapten-specific antibody responses observed in
CD38/ mice were apparent when the immunizing
antigen was presented in alum adjuvant but not when it was presented in
the stronger complete Freund's adjuvant, and these deficiencies were
influenced by the dose of the immunizing antigen (Figs 2 and 3). These
observations are consistent with our recent in vitro findings that
coengagement of CD38 and the BCR can lower the amount of antigen
receptor crosslinking required to signal through the BCR.30
Based on these data we would propose that, at least in response to the
TD antigens tested, CD38 deficiency leads to a reduced sensitivity to
signals delivered through the BCR, resulting in a requirement for a
higher antigen dose to induce B-cell activation. The fact that
CD38/ mice mounted normal hapten-specific
antibody responses to TD antigens in Freund's adjuvant (Fig 2 and data
not shown), and to the TI-2 antigen NP27-Ficoll (Fig 4B) is
also consistent with this hypothesis. B cells that have been activated
by strong adjuvants or by antigens capable of extensive BCR
crosslinking, such as polysaccharide TI-2 antigens, may not require
additional CD38-mediated costimulatory signals. If this hypothesis is
correct, one might expect that CD38-deficient mice should mount normal
antigen-specific responses to all antigens that induce extensive BCR
crosslinking and activation. However, immunization of
CD38/ mice with a second TI-2 antigen,
13 dextran, resulted in an augmented antigen-specific
response (Fig 4A). One striking difference between the immune response
to 13 dextran, and the immune response to TD antigens or
the TI-2 antigen NP27-Ficoll is that the response to
13 dextran is mediated by B-1 B cells, whereas immune
responses to the other antigens are controlled by conventional B
cells.54,55 Therefore, it is possible that CD38 performs
functionally distinct roles on conventional versus B-1 B cells. For
example, on conventional B cells CD38 may function to augment signaling
through the BCR, whereas on B-1 B cells the function of CD38 may be to
downmodulate signals through this same receptor complex. In vitro
experiments on mature splenic B cells have in fact shown that while
coengagement of CD38 and the BCR can augment B-cell activation,
independent ligation of these receptors results in a depressed
response.30,31 In addition, CD38-mediated signaling can
result in different functional responses on conventional and B-1 B
cells, as evidenced by the fact that, although anti-CD38 antibodies
provide a potent growth costimulatory signal for conventional B cells,
this is not the case for B-1 B cells.56 B-1 B cells also
fail to proliferate in response to stimulation through the
BCR,56,57 suggesting that signaling through CD38 and the
BCR may be coordinately controlled on B-1 B cells.
In any case, delineating the events associated with CD38-mediated
B-cell signaling will be critical to understanding the mechanism(s) by
which CD38 influences B lymphocyte responses. A number of laboratories have now shown that engagement of CD38 by specific antibodies can
induce tyrosine phosphorylation and activation of intracellular signal
transduction mediators.33-35 Interestingly, many of the proteins that are phosphorylated upon CD38 ligation are also
phosphorylated after BCR engagement,33,36,58-60 suggesting
that there may be extensive crosstalk between CD38 and the BCR. Of
particular interest is the recent finding of Kitanaka et
al,36 that ligation of CD38 on human immature B cells
induces the tyrosine phosphorylation of CD19 and the subsequent
association of CD19 with lyn and PI3-kinase. Based on these
observations, it was suggested that CD19 may function as a component of
the CD38-mediated signaling pathway, providing CD38 with access to
intracellular tyrosine kinase cascades. If this hypothesis is correct,
it suggests that CD38 may modulate mature B lymphocyte responses by
influencing the signaling pathway of a second B-cell coreceptor such as
CD19. The fact that CD19/ mice had a similar
but more profound impairment in humoral immune responsiveness than that
observed in CD38/ mice would be supportive of
this notion.61,62
Because antibody responses to both TD and TI-2 antigens result from
complex interactions between multiple cell types, many of which express
CD38, further experiments will be required to delineate the exact
cellular mechanism(s) responsible for the impaired humoral immunity in
these mice. Although there is strong supporting evidence that the
compromised immune responses observed in
CD38/ mice results from a loss of
CD38-mediated BCR coreceptor activity, it is also possible that these
compromised responses result from altered T-cell and/or APC
function. The fact that CD38-mediated signaling can induce cytokine
production by human peripheral blood mononuclear cells,27
enhance murine macrophage APC function (N. Solvason et al, manuscript
in preparation), and induce the activation of tyrosine kinases normally
associated with the TCR35 is at least suggestive of a role
for CD38 in modulating additional responses important for TD and TI-2
antibody responses. Whether CD38 might serve an analogous function on T
cells as it does on B cells, and act as a coreceptor for signaling
through the TCR remains to be determined. The fact that the
immunoglobulin isotypes most severely affected in the TD responses of
these mice (ie, IgG1 and IgE) are those normally supported by the Th2
subpopulation of helper T lymphocytes63 might also suggest
that the observed phenotype in these mice can be explained by a failure
to induce or maintain antigen-specific Th2 cell responses. However,
data from preliminary experiments has shown that
CD38/ mice produced normal responses
following sensitization with either Nippostrongylus
brasiliensis or Aspergillus fumigatus (data not shown),
responses that are normally Th2 T-helper-cell mediated. Alternatively,
it remains a possibility that the skewing towards deficiencies in
antigen-specific IgG1 and IgE isotype responses may simply reflect the
dominance of these isotypes after immunization with alum adjuvant.
Although our study clearly shows that a loss of CD38 impacts on immune
responses to experimental antigens, it is unclear whether this defect
is caused by a loss of CD38-associated ectoenzyme activity or to some
other function mediated by CD38. Determining the relationship between
the enzymatic activities, and the in vivo functional activities of CD38
will be one focus of future studies. It is also clear from our data
that CD38 is the primary, if not the only, NAD+
glycohydrolase in the liver and brain of mice. Thus, whereas our
studies have focused primarily on the role of CD38 in hematopoiesis and
immune regulation, it will also be of interest to evaluate CD38-deficient mice with respect to the physiological functions of
other organ systems that express CD38 enzymatic activity, such as the
pancreas, liver, and brain.
| |
FOOTNOTES |
Submitted November 19, 1997;
accepted April 15, 1998.
DNAX Research Institute is fully funded by Schering Plough Corporation.
Address reprint requests to Debra A. Cockayne, PhD, Center for
Biological Research, Neurobiology Unit, Roche Bioscience, 3401 Hillview
Ave, Palo Alto, CA 94304; e-mail: debra.cockayne{at}roche.com.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge Tom McNeil for microinjection and blastocyst
implantation, Drs Klaus Rajewsky and Werner Müller for the
NP20-CG and NP27-Ficoll reagents, Dr John
Kearney for the 13 dextran reagents, Dr Robert Coffman for
the TNP5-KLH reagents, and Nisha Kabra for her work on the
construction of the CD38 gene-targeting vector.
| |
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Y. Sasaki, B. P. S. Vohra, F. E. Lund, and J. Milbrandt
Nicotinamide Mononucleotide Adenylyl Transferase-Mediated Axonal Protection Requires Enzymatic Activity But Not Increased Levels of Neuronal Nicotinamide Adenine Dinucleotide
J. Neurosci.,
April 29, 2009;
29(17):
5525 - 5535.
[Abstract]
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F. Scheuplein, N. Schwarz, S. Adriouch, C. Krebs, P. Bannas, B. Rissiek, M. Seman, F. Haag, and F. Koch-Nolte
NAD+ and ATP Released from Injured Cells Induce P2X7-Dependent Shedding of CD62L and Externalization of Phosphatidylserine by Murine T Cells
J. Immunol.,
March 1, 2009;
182(5):
2898 - 2908.
[Abstract]
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F. Malavasi, S. Deaglio, A. Funaro, E. Ferrero, A. L. Horenstein, E. Ortolan, T. Vaisitti, and S. Aydin
Evolution and Function of the ADP Ribosyl Cyclase/CD38 Gene Family in Physiology and Pathology
Physiol Rev,
July 1, 2008;
88(3):
841 - 886.
[Abstract]
[Full Text]
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L. Mayo, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, M.-J. Moutin, F. E. Lund, and R. Stein
Dual Role of CD38 in Microglial Activation and Activation-Induced Cell Death
J. Immunol.,
July 1, 2008;
181(1):
92 - 103.
[Abstract]
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J. C. Rodriguez-Alba, M. E. Moreno-Garcia, C. Sandoval-Montes, V. H. Rosales-Garcia, and L. Santos-Argumedo
CD38 induces differentiation of immature transitional 2 B lymphocytes in the spleen
Blood,
April 1, 2008;
111(7):
3644 - 3652.
[Abstract]
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A. G. P. Guedes, J. A. Jude, J. Paulin, H. Kita, F. E. Lund, and M. S. Kannan
Role of CD38 in TNF-{alpha}-induced airway hyperresponsiveness
Am J Physiol Lung Cell Mol Physiol,
February 1, 2008;
294(2):
L290 - L299.
[Abstract]
[Full Text]
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S. Partida-Sanchez, A. Gasser, R. Fliegert, C. C. Siebrands, W. Dammermann, G. Shi, B. J. Mousseau, A. Sumoza-Toledo, H. Bhagat, T. F. Walseth, et al.
Chemotaxis of Mouse Bone Marrow Neutrophils and Dendritic Cells Is Controlled by ADP-Ribose, the Major Product Generated by the CD38 Enzyme Reaction
J. Immunol.,
December 1, 2007;
179(11):
7827 - 7839.
[Abstract]
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F. Koch-Nolte, J. Reyelt, B. Schossow, N. Schwarz, F. Scheuplein, S. Rothenburg, F. Haag, V. Alzogaray, A. Cauerhff, and F. A. Goldbaum
Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo
FASEB J,
November 1, 2007;
21(13):
3490 - 3498.
[Abstract]
[Full Text]
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S. Adriouch, S. Hubert, S. Pechberty, F. Koch-Nolte, F. Haag, and M. Seman
NAD+ Released during Inflammation Participates in T Cell Homeostasis by Inducing ART2-Mediated Death of Naive T Cells In Vivo
J. Immunol.,
July 1, 2007;
179(1):
186 - 194.
[Abstract]
[Full Text]
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J. Iqbal and M. Zaidi
CD38 is required for priming by TNF-{alpha}: a mechanism for extracellular coordination of cell fate
Am J Physiol Renal Physiol,
April 1, 2007;
292(4):
F1283 - F1290.
[Abstract]
[Full Text]
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J. D. Johnson, E. L. Ford, E. Bernal-Mizrachi, K. L. Kusser, D. S. Luciani, Z. Han, H. Tran, T. D. Randall, F. E. Lund, and K. S. Polonsky
Suppressed insulin signaling and increased apoptosis in CD38-null islets.
Diabetes,
October 1, 2006;
55(10):
2737 - 2746.
[Abstract]
[Full Text]
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Y.-G. Chen, J. Chen, M. A. Osborne, H. D. Chapman, G. S. Besra, S. A. Porcelli, E. H. Leiter, S. B. Wilson, and D. V. Serreze
CD38 Is Required for the Peripheral Survival of Immunotolerogenic CD4+ Invariant NK T Cells in Nonobese Diabetic Mice.
J. Immunol.,
September 1, 2006;
177(5):
2939 - 2947.
[Abstract]
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J. Chen, Y.-G. Chen, P. C. Reifsnyder, W. H. Schott, C.-H. Lee, M. Osborne, F. Scheuplein, F. Haag, F. Koch-Nolte, D. V. Serreze, et al.
Targeted Disruption of CD38 Accelerates Autoimmune Diabetes in NOD/Lt Mice by Enhancing Autoimmunity in an ADP-Ribosyltransferase 2-Dependent Fashion.
J. Immunol.,
April 15, 2006;
176(8):
4590 - 4599.
[Abstract]
[Full Text]
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Q. Chen and A. C. Ross
Inaugural Article: Vitamin A and immune function: Retinoic acid modulates population dynamics in antigen receptor and CD38-stimulated splenic B cells
PNAS,
October 4, 2005;
102(40):
14142 - 14149.
[Abstract]
[Full Text]
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C. Sandoval-Montes and L. Santos-Argumedo
CD38 is expressed selectively during the activation of a subset of mature T cells with reduced proliferation but improved potential to produce cytokines
J. Leukoc. Biol.,
April 1, 2005;
77(4):
513 - 521.
[Abstract]
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C. Krebs, S. Adriouch, F. Braasch, W. Koestner, E. H. Leiter, M. Seman, F. E. Lund, N. Oppenheimer, F. Haag, and F. Koch-Nolte
CD38 Controls ADP-Ribosyltransferase-2-Catalyzed ADP-Ribosylation of T Cell Surface Proteins
J. Immunol.,
March 15, 2005;
174(6):
3298 - 3305.
[Abstract]
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D. A. Deshpande, T. A. White, A. G. P. Guedes, C. Milla, T. F. Walseth, F. E. Lund, and M. S. Kannan
Altered Airway Responsiveness in CD38-Deficient Mice
Am. J. Respir. Cell Mol. Biol.,
February 1, 2005;
32(2):
149 - 156.
[Abstract]
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S. Partida-Sanchez, P. Iribarren, M. E. Moreno-Garcia, J.-L. Gao, P. M. Murphy, N. Oppenheimer, J. M. Wang, and F. E. Lund
Chemotaxis and Calcium Responses of Phagocytes to Formyl Peptide Receptor Ligands Is Differentially Regulated by Cyclic ADP Ribose
J. Immunol.,
February 1, 2004;
172(3):
1896 - 1906.
[Abstract]
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C. Ceni, H. Muller-Steffner, F. Lund, N. Pochon, A. Schweitzer, M. De Waard, F. Schuber, M. Villaz, and M.-J. Moutin
Evidence for an Intracellular ADP-ribosyl Cyclase/NAD+-glycohydrolase in Brain from CD38-deficient Mice
J. Biol. Chem.,
October 17, 2003;
278(42):
40670 - 40678.
[Abstract]
[Full Text]
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L. SUN, J. IQBAL, S. DOLGILEVICH, T. YUEN, X.-B. WU, B. S. MOONGA, O. A. ADEBANJO, P. J. R. BEVIS, F. LUND, C. L.-H. HUANG, et al.
Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption
FASEB J,
March 1, 2003;
17(3):
369 - 375.
[Abstract]
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J. Kralovicova, L. Hammarstrom, A. Plebani, A. D. B. Webster, and I. Vorechovsky
Fine-Scale Mapping at IGAD1 and Genome-Wide Genetic Linkage Analysis Implicate HLA-DQ/DR as a Major Susceptibility Locus in Selective IgA Deficiency and Common Variable Immunodeficiency
J. Immunol.,
March 1, 2003;
170(5):
2765 - 2775.
[Abstract]
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W. Ohlrogge, F. Haag, J. Lohler, M. Seman, D. R. Littman, N. Killeen, and F. Koch-Nolte
Generation and Characterization of Ecto-ADP-Ribosyltransferase ART2.1/ART2.2-Deficient Mice
Mol. Cell. Biol.,
November 1, 2002;
22(21):
7535 - 7542.
[Abstract]
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F. Tajima, T. Deguchi, J. H. Laver, H. Zeng, and M. Ogawa
Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells
Blood,
May 1, 2001;
97(9):
2618 - 2624.
[Abstract]
[Full Text]
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S. Bergthorsdottir, A. Gallagher, S. Jainandunsing, D. Cockayne, J. Sutton, T. Leanderson, and D. Gray
Signals That Initiate Somatic Hypermutation of B Cells In Vitro
J. Immunol.,
February 15, 2001;
166(4):
2228 - 2234.
[Abstract]
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S. Kahl, M. Nissen, R. Girisch, T. Duffy, E. H. Leiter, F. Haag, and F. Koch-Nolte
Metalloprotease-Mediated Shedding of Enzymatically Active Mouse ecto-ADP-ribosyltransferase ART2.2 Upon T Cell Activation
J. Immunol.,
October 15, 2000;
165(8):
4463 - 4469.
[Abstract]
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F. G. S. de Toledo, J. Cheng, M. Liang, E. N. Chini, and T. P. Dousa
ADP-Ribosyl Cyclase in Rat Vascular Smooth Muscle Cells : Properties and Regulation
Circ. Res.,
June 9, 2000;
86(11):
1153 - 1159.
[Abstract]
[Full Text]
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M. PODESTÀ, E. ZOCCHI, A. PITTO, C. USAI, L. FRANCO, S. BRUZZONE, L. GUIDA, A. BACIGALUPO, D. T. SCADDEN, T. F. WALSETH, et al.
Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors
FASEB J,
April 1, 2000;
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[Abstract]
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E. Todisco, T. Suzuki, K. Srivannaboon, E. Coustan-Smith, S. C. Raimondi, F. G. Behm, A. Kitanaka, and D. Campana
CD38 ligation inhibits normal and leukemic myelopoiesis
Blood,
January 15, 2000;
95(2):
535 - 542.
[Abstract]
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F. E. Lund, H. M. Muller-Steffner, N. Yu, C. D. Stout, F. Schuber, and M. C. Howard
CD38 Signaling in B Lymphocytes Is Controlled by Its Ectodomain but Occurs Independently of Enzymatically Generated ADP-Ribose or Cyclic ADP-Ribose
J. Immunol.,
March 1, 1999;
162(5):
2693 - 2702.
[Abstract]
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I. Kato, Y. Yamamoto, M. Fujimura, N. Noguchi, S. Takasawa, and H. Okamoto
CD38 Disruption Impairs Glucose-induced Increases in Cyclic ADP-ribose, [Ca2+]i, and Insulin Secretion
J. Biol. Chem.,
January 22, 1999;
274(4):
1869 - 1872.
[Abstract]
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K. M. Khoo, M.-K. Han, J. B. Park, S. W. Chae, U.-H. Kim, H. C. Lee, B. H. Bay, and C. F. Chang
Localization of the Cyclic ADP-ribose-dependent Calcium Signaling Pathway in Hepatocyte Nucleus
J. Biol. Chem.,
August 4, 2000;
275(32):
24807 - 24817.
[Abstract]
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C. Munshi, R. Aarhus, R. Graeff, T. F. Walseth, D. Levitt, and H. C. Lee
Identification of the Enzymatic Active Site of CD38 by Site-directed Mutagenesis
J. Biol. Chem.,
July 7, 2000;
275(28):
21566 - 21571.
[Abstract]
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Abstract
Introduction
Abstract