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: email@example.com
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