Release of Calcium from Inositol 1,4,5-Trisphosphate
Receptor-Regulated Stores by HIV-1 Tat Regulates TNF-?
Production in Human Macrophages1
Michael Mayne,* Clark P. Holden,* Avindra Nath,†and Jonathan D. Geiger2*
HIV-1 protein Tat is neurotoxic and increases macrophage and microglia production of TNF-?, a cytopathic cytokine linked to
the neuropathogenesis of HIV dementia. Others have shown that intracellular calcium regulates TNF-? production in macro-
phages, and we have shown that Tat releases calcium from inositol 1,4,5-trisphosphate (IP3) receptor-regulated stores in neurons
and astrocytes. Accordingly, we tested the hypothesis that Tat-induced TNF-? production was dependent on the release of
intracellular calcium from IP3-regulated calcium stores in primary macrophages. We found that Tat transiently and dose-de-
pendently increased levels of intracellular calcium and that this increase was blocked by xestospongin C, pertussis toxin, and by
phospholipase C and type 1 protein kinase C inhibitors but not by protein kinase A or phospholipase A2inhibitors. Xestospongin
C, BAPTA-AM, U73122, and bisindolylmalemide significantly inhibited Tat-induced TNF-? production. These results demon-
strate that in macrophages, Tat-induced release of calcium from IP3-sensitive intracellular stores and activation of nonconven-
tional PKC isoforms play an important role in Tat-induced TNF-? production. The Journal of Immunology, 2000, 164: 6538–
ical symptoms of HIV dementia, little is known about the molec-
ular mechanisms that regulate its development and progression.
Macrophages and microglia are productively infected with HIV-1
(2–4) and shed intact HIV virions and viral proteins (including
Tat) that can act directly on adjacent cells to cause increased levels
of intracellular calcium ([Ca2?]i)3(5), neurotoxicity (6–8), and
subsequent neurodegeneration (9).
The HIV-1 protein Tat may be particularly important in HIV-
associated neurodegeneration because of the following findings.
HIV-1 Tat protein (9) and transcripts (10) are present in autopsy
brain samples from HIV-infected patients with dementia. Tat is
released from HIV-infected cells (11, 12). Primary neurons and
astrocytes exposed to Tat rapidly release calcium from inositol
1,4,5-trisphosphate (IP3)-regulated pools and, subsequent to this
release of calcium, extracellular calcium that enters the cell leads
atients with HIV-1 dementia suffer multiple cognitive and
behavioral deficits and usually only survive a few months
following onset (1). Despite identifying characteristic clin-
to calcium dysregulation and neuron cell death (5, 9, 13). Tat ac-
tivates primary astrocytes, peripheral blood macrophages, and mi-
croglia to produce proinflammatory cytokines including IL-1,
IL-6, and TNF-? (14–16), and even a transient exposure of mono-
cytic and glial cells to Tat increases cytokine production (15, 17).
A neutralizing Ab to TNF-? blocks Tat-induced neurotoxicity (8).
Together, these results strongly suggest that Tat protein can acti-
vate calcium mobilization in multiple cell types within the brain
and concurrently induce proinflammatory cytokine production.
We reported recently that Tat induces IP3-regulated calcium re-
lease in neurons and astrocytes and that this increase leads to a
dysregulation in [Ca2?]iand neurotoxicity (5). Others have shown
that intracellular stores of calcium play important roles in regulat-
ing TNF-? production in primary human macrophages (18–22).
Because TNF-? has been implicated as a pathogenic factor in HIV
disease (23), is elevated in the brains of HIV-infected patients (24),
and Tat has been shown to elevate TNF-? production in mono-
cytes (14, 25), these events may lead to highly activated microglia
and macrophages in the brain, an event that correlates with the
clinical symptoms of AIDS dementia (3). Thus, it is important to
identify the cellular mechanisms that mediate these pathways. Ac-
cordingly, we tested the hypothesis that Tat-induced TNF-? pro-
duction involved the release of [Ca2?]ifrom IP3-regulated calcium
stores in primary macrophages. Here, we report that exposure of
human primary macrophages to Tat protein induces a rapid and
dose-dependent release of calcium from IP3-regulated intracellular
stores and that Tat-induced TNF-? production was dependent, at
least in part, on the release of calcium from those stores.
Materials and Methods
Chemicals and recombinant Tat
ATP, caffeine, [1-(5-isoquinolinesulfonyl)-2-methylpiperazine] hydrochlo-
ride (H7), [N-2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfon-
amide hydrochloride (H89), EDTA, EGTA, pertussis toxin (PT), cholera toxin
(CT), citicoline (CIT), and 4-bromophenyl bromide (BPB) were purchased
from Sigma (St. Louis, MO). Xestospongin C (XsC) and bisinolylmaleimide
*Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg,
Manitoba, Canada; and†Departments of Neurology, Microbiology, and Immunology,
University of Kentucky, Lexington, KY 40506
Received for publication February 8, 2000. Accepted for publication April 3, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Support for this research was provided by the Medical Research Council of Canada
and the National Institutes of Health. M.M. was supported by an AstraZeneca/MRC/
Alzheimer’s Society of Canada Post Doctoral Operating Fellowship. C.P.H. was sup-
ported by a Medical Research Council Postdoctoral Fellowship.
2Address correspondence and reprint requests to Dr. Jonathan D. Geiger, Department
of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba,
Winnipeg, Manitoba, R3E 0T6, Canada. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: [Ca2?]i, intracellular calcium; IP3, inositol 1,4,5-
trisphosphate; Bis, bisinolylmaleimide; BPB, 4-bromophenyl bromide; CIT, citicoline;
CT, cholera toxin; fura 2-AM, fura-2-acetoxymethyl ester; H7, [1-(5-isoquinolinesulfo-
nyl)-2-methylpiperazine] hydrochloride; H89, [N-2-((p-bromocinnamyl)amino)ethyl]-5-
isoquinolinesulfonamide hydrochloride; PT, pertussis toxin; PKA, protein kinase A; PLC,
phospholipase C; PKC, protein kinase C; XsC, xestospongin C; PLA2, phospholipase A2;
BAPTA-AM, bis(2-aminophenoxy)ethane-N,N,N?,N?-tetraacetate acetoxymethyl ester.
Copyright © 2000 by The American Association of Immunologists0022-1767/00/$02.00
(Bis) were purchased from Calbiochem (San Diego, CA). Fura-2-acetoxym-
ethyl ester (fura 2-AM) and bis(2-aminophenoxy)ethane-N,N,N?,N?-tetraac-
etate acetoxymethyl ester (BAPTA-AM) were obtained from Molecular
Probes (Eugene, OR). The phospholipase C (PLC) inhibitor U73122 was pur-
chased from Research Biochemicals (Natick, MA). Anti-ferretin Abs were
purchased from Transduction Laboratories (Lexington, KY). Tat1–72was pre-
pared and purified as described previously (26) and its biological activity was
confirmed by activation of ?-galactosidase in transfected HeLa cells (AIDS
Repository, National Institutes of Health). Tat protein was lyophilized and
stored at ?80°C. Freshly thawed Tat was used in all experiments.
Preparation of primary macrophage cultures
Human PBMC and macrophages were purified from whole blood obtained
from healthy volunteers (27), and cells were cultured at a density of 2.0 ?
105cells/ml for 7 days in RPMI 1640 supplemented with 10% FBS and
antibiotics. All cells were maintained at 37°C in a humidified growth
chamber supplemented with 5% CO2.
Levels of [Ca2?]i
[Ca2?]iwere determined using the Ca2?-specific fluorescent probe fura
2-AM as described previously (28). Macrophages were excited at 340 and
380 nm, and emission was recorded at 510 nm with a video-based universal
imaging system (EMPIX, Missassauga, ON). Rmax/Rminratios were con-
verted to nanomolar [Ca2?]i(29), and pressure application of Tat and im-
age acquisition were performed as described previously (5). Peak increases
of [Ca2?]iwere determined by subtracting baseline [Ca2?]ifrom the max-
imum [Ca2?]iachieved during a 15-min period following Tat application.
Macrophage TNF-? production following Tat application
Primary macrophages were treated with Tat (100 nM) and incubated for 4 h
at 37°C. Supernatants were collected, centrifuged at 700 ? g for 5 min, and
analyzed for TNF-? by ELISA (30). For positive controls, cells were
treated with 10 ng/ml LPS (LPS, Escherichia coli type 055:B5; Sigma) for
4 h. In experiments where antagonists or inhibitors were used, these agents
were added 30 min before stimulation with Tat. Following a 4-h incuba-
tion, cell culture supernatants were collected and assayed for TNF-? abun-
dance by ELISA.
Significant differences between groups were determined by one-way
ANOVA with Tukey’s posthoc comparisons. For all tests, statistical sig-
nificance was considered to be at the p ? 0.01 level (Instat2; Graphpad
Software, San Diego, CA).
Dose-related effects of HIV-1 Tat on macrophage [Ca2?]i
We reported previously that Tat application to cultured human
astrocytes and neurons increased [Ca2?]i(5). We found here that
Tat (100 nM) applied to highly purified macrophage cultures
(?97% of the cells in the primary culture were immunoreactive
with anti-ferritin Ab; data not shown) increased significantly (p ?
0.01) [Ca2?]ifrom basal levels of 129 ? 1 nM (n ? 718; data not
shown) to maximum levels of 793 ? 69 nM (n ? 42) (Fig. 1).
These increases in [Ca2?]iwere dose related with an apparent
EC50value of 6.0 ? 0.3 nM. Independent of the experimental
conditions outlined below, agonist or antagonist treatments did not
reduce cell viability as determined by trypan blue exclusion (data
Tat releases calcium from IP3-regulated stores via a PT-
sensitive PLC-mediated pathway
In our previous work with cultured human neurons and astrocytes,
we reported that Tat-induced initial transient increases in [Ca2?]i
were due to release from IP3-regulated stores (5). Similarly, in
primary macrophages, Tat induced a sharp and transient increase
in [Ca2?]i(Fig. 2A). To determine the source of this release, we
first used the specific inhibitor of IP3-dependent calcium release,
XsC, and found that it significantly reduced (p ? 0.01) Tat-in-
duced calcium transients by 91 ? 2% (Fig. 2B; n ? 20). As a
positive control, XsC significantly blocked (p ? 0.001) ATP-in-
duced release of [Ca2?]iby 90 ? 7% (Fig. 2B; n ? 28); ATP is a
well-characterized releaser of calcium from IP3receptor-regulated
stores. Caffeine (20 mM; n ? 18) did not increase [Ca2?]i, and
ryanodine (10 ?M; n ? 25) did not affect Tat-induced increases in
[Ca2?]i(data not shown). These latter results indicate that calcium
in these cells was not being released from caffeine-sensitive ryan-
odine receptor-regulated intracellular stores. When Tat was ap-
plied to macrophages in calcium-free buffer, increases in [Ca2?]i
were not significantly different from increases observed in cells
bathed in media containing calcium (Fig. 2B).
To determine signaling events mediating Tat-induced release of
calcium from IP3-regulated stores, we used selective inhibitors that
target potential activators and effectors of PLC. PT (100 ng/ml)
significantly decreased (p ? 0.001) Tat-induced increases in
[Ca2?]iby 95 ? 1% (Fig. 2C; n ? 50). CT (100 ng/ml) did not
reduce Tat-induced increases in [Ca2?]i(Fig. 2C; n ? 44). The
PLC inhibitor U73122 significantly reduced (p ? 0.01) Tat-in-
duced increases in [Ca2?]iby 90 ? 3% (n ? 23). Because phos-
pholipase A2(PLA2) inhibition has been shown to be dependent on
PLC activity (31, 32), we tested the involvement of PLA2in Tat-
induced increases in [Ca2?]i. The selective PLA2inhibitor citico-
line (10 ?M) did not inhibit Tat-induced increases in [Ca2?]i(Fig.
2C; n ? 9). However, inhibition of protein kinase C (PKC) type 1
isoforms using H7 or Bis significantly decreased (p ? 0.01) Tat-
induced increases in [Ca2?]iby 70 ? 8% (n ? 59) and 76 ? 11%
(n ? 41), respectively (Fig. 2C). Inhibition of protein kinase A
(PKA) using H89 (n ? 27) did not attenuate Tat-induced increases
in [Ca2?]i(Fig. 2C).
Tat-induced increases in TNF-? involved rapid calcium release
from IP3-sensitive stores
Because TNF-? production has been shown to be dependent on
increases in [Ca2?]i(18–22), we hypothesized that transient in-
creases in [Ca2?]iinduced by Tat may increase the levels of Tat-
induced TNF-? production (14, 25). LPS and Tat significantly in-
creased (p ? 0.001) the levels of TNF-? (Fig. 3A). When LPS or
Tat was applied under calcium-free conditions, TNF-? production
was inhibited significantly (p ? 0.01) by 72 ? 6% and 78 ? 11%,
respectively, compared with LPS or Tat applied under calcium-
containing conditions (Fig. 3A). In the absence of extracellular
calcium, Tat-induced increases in TNF-? were significantly inhib-
ited (p ? 0.001) with BAPTA-AM by 86 ? 5% and with XsC by
64 ? 7% (Fig. 3A). In the presence of calcium, neither
human macrophages. Tat pressure-applied at concentrations ranging from
100 pM to 100 nM (3 ? 100 ms, 5 psi) induced significant (p ? 0.05)
increases of [Ca2?]iranging from 134 ? 41 to 793 ? 69 nM.
HIV-1 Tat dose-dependently increased [Ca2?]iin primary
6539The Journal of Immunology
BAPTA-AM (1 or 10 ?M) nor XsC (1 or 10 ?M) significantly
inhibited TNF-? production (data not shown).
We showed previously in monocytes that U73122 inhibited Tat-
induced TNF-? production by ?80% (14). In confirmation, in pri-
mary macrophages, U73122 significantly inhibited (p ? 0.001)
Tat-induced increases in TNF-? by 95 ? 2% (Fig. 3B). In attempt-
ing to identify G proteins involved in this PLC-mediated response,
we conducted the following experiments and found that PT, an
inhibitor of Giproteins, CT, an activator of Gsproteins, and BPB
or CIT, specific inhibitors of PLA2, did not inhibit Tat-induced
TNF-? production (Fig. 3B). Because inhibition of PLC blocked
calcium pools in a PT-sensitive, PLC-dependent pathway. A, A typical
increase of [Ca2?]ifrom a cultured human macrophage following pressure
application (arrow) of HIV-1 Tat protein (100 nM, 3 ? 100 ms, 5 psi). In
the presence of 1 ?M XsC, Tat-induced increases of [Ca2?]iwere de-
creased (second arrow). Pressure application of Tat following the removal
of XsC typically increased [Ca2?]i(third arrow). B, Effect of Tat (100 nM)
and, as a positive control, ATP (100 ?M) on the release of [Ca2?]ifrom
primary human macrophages. Normal extracellular calcium levels (f) or
removal of extracellular calcium (?) did not alter Tat- or ATP-induced
increases of [Ca2?]i. XsC (1 ?M) inhibited Tat- and ATP-induced in-
creases of [Ca2?]i. C, Tat-induced increases of [Ca2?]iin macrophages
were decreased significantly in the presence of PT (100 ng/ml), U73122
(U73) (10 ?M), H7 (10 ?M), and Bis (50 nM). CT (100 ng/ml), CIT (10
?M), and the PKA inhibitor H89 (1 ?M) were ineffective. Macrophages
were cultured in the presence of extracellular calcium (f). a, p ? 0.01,
differed significantly from macrophages treated with Tat (100 nM). b, p ?
0.001, differed significantly from macrophages treated with Tat (100 nM)
in the presence or absence of extracellular calcium. c, p ? 0.001, differed
significantly from macrophages treated with ATP (100 ?M).
HIV-1 Tat increased transiently [Ca2?]ivia IP3-regulated
[Ca2?]i. A, Macrophages cultured in the absence of extracellular calcium
(?) produced significantly lower levels of TNF-? in response to Tat (100
nM) or LPS (10 ng/ml) treatment compared with macrophages cultured in
the presence of extracellular calcium (f). In the absence of extracellular
calcium macrophages treated with XsC (10 ?M) or BAPTA-AM (10 ?M)
produced significantly lower levels of TNF-? compared with cells treated
with Tat alone. B, Macrophages cultured in the presence of extracellular
calcium (BKD) were treated with PT, CT, U73122 (U73), CIT, and BPB
and subsequently stimulated with Tat. Only inhibition of PLC (U73122)
reduced Tat-induced TNF-? production. C, Macrophages were treated with
the selective PKC type 1 isoform inhibitors H7 (1 ?M) or Bis (50 nM or
6 ?M) before treatment with Tat (100 nM). Bis (6 ?M) inhibited Tat-
induced TNF-? production. TNF-? release from primary macrophages av-
eraged 7502 ? 245 ng/106cells/ml 4 h following the application of Tat. All
experiments were conducted in quadruplicate. a, p ? 0.01, differed signif-
icantly from macrophages cultured in extracellular calcium and treated
with LPS (10 ng/ml). b, p ? 0.01, differed significantly from macrophages
cultured in extracellular calcium and treated with Tat (100 nM). c, p ?
0.01, differed significantly from macrophages cultured in the absence of
extracellular calcium and treated with Tat (100 nM). d, p ? 0.001, differed
significantly from macrophages cultured in the presence of extracellular
calcium and treated with Tat (100 nM).
Tat-induced TNF-? production is regulated by elevations in
6540INVOLVEMENT OF IP3IN TAT-INDUCED TNF-? PRODUCTION
Tat-induced calcium release from IP3receptor-regulated stores and
TNF-? production, we determined the extent to which PKC-me-
diated Tat-induced TNF-? production. In agreement with our pre-
vious work in THP-1 cells (14), H7 (an inhibitor of PKC type 1
isoforms) did not inhibit Tat-induced TNF-? production (Fig. 3C).
Bis, an inhibitor of PKC type 1 isoforms at a low concentration of
50 nM (33), did not reduce TNF-? production in primary macro-
phages exposed to 100 nM Tat (Fig. 3C). However, at a higher
concentration of 6 ?M, Bis significantly inhibited (p ? 0.01) Tat-
induced increases in TNF-? production by 53 ? 8% suggesting
that nonconventional PKC isoforms mediated Tat-induced in-
creases in TNF-? production (34).
Apoptotic and/or necrotic neuronal cell death associated with HIV
dementia appears to be caused by indirect mechanisms induced by
HIV proteins including increases in levels of [Ca2?]i(5) and proin-
flammatory cytokines (14, 15). The HIV-1 protein Tat may be a
particularly important pathogenic factor in the development and
progression of dementia because Tat has been shown to increase
levels of [Ca2?]i(5, 35), increase neuronal cell death (35, 36), and
increase TNF-?, IL-1?, IL-1?, and IL-6 production (8, 14, 15).
Here, we focused our experiments to determine signaling events
through which Tat drives TNF-? production. This issue is impor-
tant because Tat protein is present in the brain of patients with HIV
dementia (36–38), Tat transcripts are elevated in brains of patients
with HIV-1 dementia and encephalitis (38), and primary macro-
phages, which can be recruited into the CNS upon activation and
from which proinflammatory cytokines are primarily released, are
activated by HIV-1 Tat (14, 15, 37).
Similar to our previous findings in neurons and astrocytes (5),
we found, in macrophages, that Tat caused rapid and transient
increases in [Ca2?]ieven in the absence of extracellular calcium.
These findings suggest that Tat-induced increases in [Ca2?]iorig-
inated from [Ca2?]istores. The calcium released by Tat originated
from IP3-regulated pools in a PT-sensitive PLC-mediated manner.
This conclusion is based on our observations that Tat-induced in-
creases in [Ca2?]iwere inhibited significantly, by XsC, a selective
inhibitor of [Ca2?]irelease channels regulated by IP3receptors, by
PT, an inhibitor of Giproteins, and by U73122, a PLC inhibitor.
Tat-induced increases in [Ca2?]iwere not due to release of [Ca2?]i
from ryanodine receptor-regulated [Ca2?]irelease channels nor the
endoplasmic reticulum-resident calcium release channels in addi-
tion to those regulated by IP3receptors, because neither caffeine
nor ryanodine affected Tat-induced increases in [Ca2?]i. In con-
trast to our observations in some neurons and astrocytes (5), Tat
did not induce sustained increases in [Ca2?]iin any of the mac-
rophages examined. The increase in [Ca2?]iby Tat that originated
primarily from intracellular stores was dependent on PKC type 1
and nonconventional PKC isoforms. This latter observation, in
combination with our previous results that tyrosine kinases were
involved in Tat-induced TNF-? production (14), suggests that Tat
activates multiple kinase pathways.
Several studies have demonstrated that in monocytes the release
of IP3receptor-regulated [Ca2?]istores results in increased TNF-?
production (18–22, 39). In concert with those findings, we found
that Tat-induced increases in calcium release from IP3receptor-
regulated stores are also involved in TNF-? production. Although
we did not observe any evidence for extracellular calcium influx in
primary macrophages, our results do demonstrate a role for extra-
cellular calcium, in addition to [Ca2?]i, in regulating Tat-induced
TNF-? production. This is in contrast to some of our previous
observations in neurons and astrocytes where we did observe some
evidence of extracellular calcium influx (5). The likely explanation
for not observing evidence of calcium influx in macrophages, but
yet seeing an involvement of extracellular calcium in TNF-? pro-
duction, is that the rapid and transient release of calcium from IP3
receptor-regulated stores resulted in the activation of capacitative
mechanisms and small increases in [Ca2?]ithat were not detect-
able in our system. Thus, the temporal order of signaling events
that occurs in macrophages activated by Tat to produce TNF-? are
increases in [Ca2?]ithat precede capacitative entry of extracellular
calcium. This conclusion is supported by our observations that
U73122, a selective inhibitor of PLC, reduced significantly in-
creases in [Ca2?]iand TNF-? production even in the presence of
extracellular calcium. Our conclusion that calcium release from
IP3receptor-regulated stores initiates Tat-induced production of
TNF-? before influx of extracellular calcium is supported further
by our findings that the L-type calcium channel inhibitors nimo-
dipine and nicardipine did not significantly reduce Tat-induced
increases in TNF-? production (data not shown). Further, it was
reported recently that nimodipine did not afford clinical benefit of
HIV-1 dementia (40). Thus, in primary macrophages, Tat-induced
increases in [Ca2?]iresulting primarily from release from IP3re-
ceptor-regulated stores may represent the seminal signaling event
required for the production of TNF-?.
Although our results demonstrated that Tat activation of PLC is
critical for increases in [Ca2?]ithat initiate TNF-? gene expres-
sion, additional signaling events are required for maximal TNF-?
production. Contrary to our finding that Tat-induced increases in
[Ca2?]iwere PT sensitive, TNF-? production was not PT sensi-
tive, suggesting that Tat activated at least two independent G pro-
tein signaling pathways. Further, our experiments indicated that
Tat-induced TNF-? production involved nonconventional PKC
isoforms. This conclusion is based on two observations. First, Bis
(6 ?M) inhibited both Tat-induced increases in [Ca2?]iand TNF-?
production, and these high concentrations of Bis inhibit noncon-
ventional PKC isoforms (34). Second, although H7, a selective
blocker of PKC type 1 isoforms, lowered Tat-induced increases in
[Ca2?]i, it did not inhibit TNF-? production. These findings are
consistent with findings that Tat activates nonconventional PKC
isoforms including ? and ? in PC12 cells (41), that HIV-1 regulates
NF-?B via PKC? in infected monocytes (42), and that multiple
signaling pathways are activated by Tat (9).
Finally, very low concentrations (EC50of 6 nM) of Tat caused
significant increases in [Ca2?]iand TNF-? production in primary
macrophages (14). Similar concentrations have been demonstrated
in serum from HIV-infected patients (43). It has also been shown
that Tat can cross the intact blood-brain barrier (44). Given that
even a transient exposure of Tat induces a rapid and sustained
production of cytokines (15), even extremely small amounts of Tat
present in an HIV-infected brain (37) may efficiently activate in-
filtrating macrophages and resident microglia to produce excessive
amounts of TNF-?. Further, because macrophages and microglia
are the most commonly infected cells in AIDS brain (2), Tat re-
lease from these cells may lead to persistent activation and exces-
sive production of multiple proinflammatory cytokines and che-
mokines that are implicated in the development and progression of
HIV dementia. We conclude that pharmacological strategies that
target the IP3pathway may be therapeutically beneficial in the
treatment of HIV dementia.
We thank registered nurses M. Dott and T. Olafson for their help in col-
lecting the blood specimens.
6541The Journal of Immunology
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6542 INVOLVEMENT OF IP3IN TAT-INDUCED TNF-? PRODUCTION