Δ-Tetrahydrocannabinol (THC) enhances lipopolysaccharide-stimulated tissue factor in human monocytes and monocyte-derived microvesicles

Abstract

Immunomodulatory effects in humans of Δ(9-)Tetrahydrocannabinol (THC), the psychoactive component of marijuana are controversial. Tissue factor (TF), the activator of the extrinsic coagulation cascade, is increased on circulating activated monocytes and is expressed on microvesicles released from activated monocytes during inflammatory conditions, which perpetuate coagulopathies in a number of diseases. In view of the increased medicinal use of marijuana, effects of THC on human monocytes and monocyte-derived microvesicles activated by lipopolysaccharide (LPS) were investigated.
Peak levels of TF procoagulant activity developed in monocytes or microvesicles 6 h following LPS treatment and were unaltered by THC. After 24 h of LPS stimulation, TF activity declined in control-treated or untreated cells and microvesicles, but persisted with THC treatment. Peak TF protein occurred within 6 h of LPS treatment independent of THC; by 24 h, TF protein declined to almost undetectable levels without THC, but was about 4-fold greater with THC. Steady-state TF mRNA levels were similar up to 2 h in the presence of LPS with or without THC, while 10-fold greater TF mRNA levels persisted over 3-24 h with THC treatment. Activation of MAPK or NF-κB pathways was unaltered by THC treatment and inflammatory cytokine IL-6 levels were unchanged. In contrast, TNF and IL-8 levels were enhanced by 20-50 %.
THC enhances TF expression in activated monocytes resulting in elevated procoagulant activity. Marijuana use could potentiate coagulopathies in individuals with chronic immune activation such as HIV-1 infection or inflammatory bowel disease.

Full text

S H O R T R E P O R T Open Access
Δ
9
-Tetrahydrocannabinol (THC) enhances
lipopolysaccharide-stimulated tissue factor
in human monocytes and monocyte-
derived microvesicles
Julie C. Williams
1
, Thomas W. Klein
2
, Bruce A. Goldberger
1
, John W. Sleasman
3
, Nigel Mackman
4
and Maureen M. Goodenow
1*
Abstract
Background: Immunomodulatory effects in humans of Δ
9−
Tetrahydrocannabinol (THC), the psychoactive component of
marijuana are controversial. Tissue factor (TF), the activator of the extrinsic coagulation cascade, is increased on circulating
activated monocytes and is expressed on microvesicles released from activated monocytes during inflammatory
conditions, which perpetuate coagulopathies in a number of diseases. In view of the increased medicinal use of
marijuana, effects of THC on human monocytes and monocyte-derived microvesicles activated by lipopolysaccharide
(LPS) were investigated.
Findings: Peak levels of TF procoagulant activity developed in monocytes or microvesicles 6 h following LPS treatment
and were unaltered by THC. After 24 h of LPS stimulation, TF activity declined in control-treated or untreated cells and
microvesicles, but persisted with THC treatment. Peak TF protein occurred within 6 h of LPS treatment independent of
THC; by 24 h, TF protein declined to almost undetectable levels without THC, but was about 4-fold greater with THC.
Steady-state TF mRNA levels were similar up to 2 h in the presence of LPS with or without THC, while 10-fold greater
TF mRNA levels persisted over 3–24 h with THC treatment. Activation of MAPK or NF-κB pathways was unaltered by
THC treatment and inflammatory cytokine IL-6 levels were unchanged. In contrast, TNF and IL-8 levels were enhanced
by 20–50 %.
Conclusions: THC enhances TF expression in activated monocytes resulting in elevated procoagulant activity.
Marijuana use could potentiate coagulopathies in individuals with chronic immune activation such as HIV-1 infection or
inflammatory bowel disease.
Keywords: Lipopolysaccharide, Marijuana, Monocyte, Tissue factor, THC
Introduction
Tissue factor (TF) is a membrane-bound protein that
initiates the extrinsic pathway of the coagulation
cascade [1]. In vitro, the signaling and kinetics of
lipopolysaccharide (LPS)-stimulated TF expression on
monocytes and microvesicles are well understood. LPS
stimulation of monocytes leads to mitogen activated
protein kinase (MAPK) and nuclear factor κB(NF-κB)
activation resulting in transcription of TF mRNA
followed by translation of TF protein [2–4]. LPS stimu-
lation increases steady state levels of TF mRNA and
protein expression, however TF is regulated post-
transcriptionally and post-translationally, resulting in a
peak expression followed by steady decline [4, 5]. TF
expression by monocytes or microvesicles in the circu-
lation is minimal under normal physiologic conditions,
while circulating monocytes perturbed by infection or
inflammation upregulate TF, and subsequently, release
TF via microvesicles [6].
* Correspondence: goodenow@ufl.edu
1
Department of Pathology, Immunology and Laboratory Medicine, College of
Medicine, University of Florida, 2033 Mowry Road, Gainesville, FL 32610-3663,
USA
Full list of author information is available at the end of the article
© 2015 Williams et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Williams et al. Journal of Inflammation (2015) 12:39
DOI 10.1186/s12950-015-0084-1

Microvesicles are 100–1000 nm membrane blebs that
are released in response to stimulation or cell death [7].
Microvesicles transport cellular signals via their cargo,
which can include microRNA, RNA, DNA or proteins
[7]. Monocyte-derived microvesicles are significant
sources of pro-coagulant activity due to expression of
TF, as well as phosphatidylserine, a cofactor for the
coagulation cascade.
Individuals with HIV-1 infection and Inflammatory Bowel
Disease (IBD) or other diseases with elevated plasma LPS
have increased TF expression on monocytes and TF
+
microvesicles [8, 9] and are at increased risk for
coagulopathies [8, 10]. Marijuana is proposed for pharma-
cological interventions for either HIV-1 infection or IBD
[11, 12]. The psychoactive component of marijuana,
Δ
9−
tetrahydrocannabinol (THC), has immunomodulatory
properties, although most studies showing that THC is
anti-inflammatory were performed in animal models,
murine cells or transformed human cell lines [13]. Human
monocytes express THC receptors [13], however the
effects of THC on human monocytes, microvesicles, and
coagulation are unknown. Here, we investigated the effects
of THC on LPS-stimulated TF expression and activity in
human monocytes and monocyte-derived microvesicles.
624
0
10
20
30
*
Hours of LPS stimulation
Microvesicle
TF activity (pg/mL)
AB
C
624
0
20
40
60
80
*
Hours of LPS st imulation
Monocyte
TF activity (pg/mL)
00.5 2 6 12 24
0.1
1
10
100
1000
10000
Vehicle
THC
Hours of LPS stimulation
TF expression
0
2
4
6
0.51 2 3 6 12 24
Hours of LPS stimulation
Relative TF expression
TF
actin
THC
LPS +
++++
Time(hrs)
+
0 2462
++++
Vehicle ++ ++
DE
Fig. 1 THC prolongs TF expression and procoagulant activity. Monocytes were treated with 30 μM THC or vehicle control for 30 min prior to the
addition of LPS (100 ng/mL) for indicated time periods. TF activity in (a) monocytes or (b) microvesicles isolated from monocyte supernatants in
(a) was measured. Graphs are results are from one donor, showing mean and standard error from 3 wells. Similar results were obtained in cells
from 3 donors. TF activity is donor dependent, and, in the absence of LPS stimulation, usually undetectable, but never greater than 1 pg/mL in
media only or THC only controls. Grey bars are LPS only, open bars are vehicle control and LPS, solid black bars are THC and LPS. * p< 0.001 via
ANOVA followed by Bonferroni post test comparing THC to vehicle. cWhole cell lysates from monocytes were analyzed by western blot for
indicated proteins. TF western blots were stripped and reprobed for actin. d,eTotal mRNA was isolated from monocytes treated with THC or
vehicle 30 min prior to LPS and analyzed for TF and GAPDH by quantitative real-time PCR. dTF versus GAPDH mRNA from one representative
donor is graphed. Data is presented as a relative fold change compared to untreated cells. eMean and standard error from at least 3 donors are
graphed relative to vehicle (dotted line at 1) at indicated time points
Williams et al. Journal of Inflammation (2015) 12:39 Page 2 of 6

Methods
Cells and reagents
Elutriated human monocytes were obtained from Dr. Mark
Wallet at the University of Florida under protocols ap-
proved by the Institutional Review Board. Monocytes were
rested overnight in Dulbecco’s Modified Eagle Medium
(DMEM) (Corning) containing 10 % human serum, Cipro-
floxacin (Corning), and Gentamicin (Sigma) prior to
addition of THC (Sigma) or ethanol vehicle control. In all
experiments, THC or vehicle alone was added 30 min prior
to stimulation with LPS from E.coli O111:B4 (Sigma).
Isolation of microvesicles and flow cytometry
Microvesicles were isolated from cell and cellular debris
free supernatants by centrifugation at 16,000xg for
15 min at 4 °C and Annexin V FITC staining observed
by flow cytometry, as previously described [14].
Tissue factor activity assay
TF procoagulant activity assay was performed as previ-
ously described [15].
Protein analysis: Western Blot and ELISA
Whole cell lysates were obtained using lysis buffer (Cell
Signaling Technology) from monocytes stimulated with
THC or vehicle 30 min prior to 100 ng/mL LPS for indi-
cated time periods. Lysates were analyzed by sodium dode-
cyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to polyvinyl difluoride (PVDF) membrane
(Bio-Rad). Membranes were probed using anti-TF antibody
(BD Biosciences) or antibodies to actin, pERK1/2, total
0
1
2
3
4
*
**
Ratio of TF to actin
0
1
2
3
4
**
Monocyte TF activity
THC (µM)
LPS
TF
actin
+
++++
1103030
A
BC
0.0
0.5
1.0
1.5
2.0
2.5
**
Microvesicle TF activity
0.0
0.5
1.0
1.5
2.0
Annexin V+ events
DE
Vehicle +
THC (µM)
LPS +++++
1103030
+
Vehicle
THC (µM)
LPS +++++
1103030
+
Vehic le
THC (µM)
LPS +++++
1103030
+
Vehicle
THC (µM)
LPS +++++
1103030
+
Vehic le
Fig. 2 THC has dose dependent effects on TF expression and activity. Monocytes were treated with THC at indicated concentrations or vehicle
control for 30 min prior to the addition of 100 ng/mL LPS for 24 h. aWhole cell lysates were analyzed by western blot for indicated proteins.
TF western blots were stripped and reprobed for actin. bDensitometry showing ratio of TF to actin across 5 donors. cMonocyte TF activity was
measured. d,eMicrovesicles were prepared from supernatants from monocytes treated as in a-cand measured for dmicrovesicle TF activity or
ecounted by flow cytometry. b-ePanels represent at least 5 donors and data are expressed relative to LPS only treatment (grey bar). Graphs
show mean and standard error. * p< 0.05, ** p< 0.01 via ANOVA followed by Bonferroni post test
Williams et al. Journal of Inflammation (2015) 12:39 Page 3 of 6

ERK1/2, phospho-p65, total p65, or IκBα(Cell Signaling
Technology). Densitometry was performed in Image J
(NIH). Band intensity ratios of TF to actin were quantified
and then normalized to LPS only treatments. ELISA for
human interleukin-6 (IL-6), tumor necrosis factor-α
(TNFα), interleukin-8 (IL-8) (BD Biosciences) were per-
formed according to manufacturer’sinstructions.
RNA isolation and quantitative real-time PCR
RNA was isolated and reverse transcribed into cDNA as
previously described [16]. Quantitative real-time PCR
(qPCR) was performed using primers and probes for TF,
IL-6, TNFα, IL-8 and gyceraldehyde 3-phosphate de-
hydrogenase (GAPDH) (IDT Technologies) using an
ABI 7500 FAST instrument.
Statistical analysis
Statistical analysis using ANOVA followed by Bonferoni
post test was performed in GraphPad Prism. A pvalue
less than 0.05 was considered statistically significant.
Results and discussion
The effect of THC pretreatment on LPS-stimulated TF
procoagulant activity of monocytes was measured over the
course of 24 h. Peak TF activity occurred after 6 h of LPS
stimulation independent of THC treatment (Fig. 1a). By
24 h, TF procoagulant activity remained elevated in THC-
treated LPS-stimulated monocytes, but declined signifi-
cantly in vehicle-treated or untreated LPS-stimulated cells
(Fig. 1a). Similar to monocyte TF activity, peak microvesicle
TFactivityoccurredby6hofLPS stimulation (Fig. 1b).
After 24 h, TF activity of microvesicles from THC-treated
cells was approximately 3-fold greater compared to vehicle-
treated or untreated LPS stimulated MVs (Fig. 1b). Al-
though the magnitude of monocyte and microvesicle
TF activity was donor dependent, THC-mediated increased
TF activity occurred with a range of 2- to 6-fold at 24 h
among donors. Results indicate that THC modulation of
TF activity in monocytes was paralleled by elevation in
microvesicle TF activity.
To confirm that TF activity was the result of elevated
TF protein expression and not dependent on a co-
factor or post-translational modification, TF protein
levels were evaluated by western blot. TF protein ap-
peared by 2 h post LPS stimulation (Fig. 1c). No differ-
ences in levels were apparent until 24 h of LPS
stimulation, when THC-treated cells had greater levels
of TF protein compared to cells treated with vehicle
control. As expected, TF protein was undetectable in
the absence of LPS stimulation (data not shown). Next,
TF mRNA levels were evaluated over 24 h by qPCR. TF
mRNA levels in THC-treated and vehicle control-
Fig. 3 No effect of THC on selected LPS-stimulated signal transduction molecules. Monocytes were stimulated with 30 μM THC or vehicle 30 min
prior to 100 ng/mL LPS for indicated time periods. aWhole cell lysates were subjected to western blot for indicated proteins. Representative blots
of one donor are shown. Similar results were obtained in 3 donors. Phospho-ERK1/2(p-ERK1/2) blot was stripped and reprobed for total ERK1/2.
Phospho-p65 (p-p65) blot was stripped and reprobed for total p65. IκBαblot was stripped and reprobed for actin. Densitometry of data from 3
donors is graphed for pERK1/2: total ERK1/2 ratio (b), p-p65: total p65 ratio (c), and IκB: actin ratio (d). Graphs are normalized to untreated samples
and depict mean and standard error for 3 donors
Williams et al. Journal of Inflammation (2015) 12:39 Page 4 of 6

treated cells remained similar until peak expression 2 h
post LPS stimulation, but were elevated in THC-treated
cells at all subsequent time points (Fig. 1d). Among 5
donors, levels of TF mRNA in the presence of THC
were approximately 2-fold higher at 3 and 6 h, increas-
ing to 4-fold higher by 12 and 24 h (Fig. 1e). Although
the magnitude of THC responses differed among
donors, all donors showed THC-mediated elevated TF
expression from 2 to 5-fold relative to control over 3 to
24 h of LPS stimulation (Fig. 1e). Findings indicate that
THC treatment prolongs levels of TF mRNA and pro-
tein, as well as TF procoagulant activity, in both mono-
cytes and monocyte-derived microvesicles.
Enhancement of monocyte TF protein levels by THC was
dose dependent with maximal production at the 30 μM
dose (Fig. 2a, b). Monocyte TF activity increased 250 % at
the 30 μM dose (Fig. 2c). Similar to monocyte TF activity,
LPS-stimulated microvesicle TF activity showed a dose
dependent THC mediated elevation (Fig. 2d). Similar
numbers of microvesicles were observed by flow cytometry
between THC- and vehicle-treated or untreated LPS-
stimulated supernatants (Fig. 2e), indicating that TF activity
increased per microvesicle rather than via increased micro-
vesicle release by THC-treated LPS-stimulated cells.
Concentrations of THC used were higher than levels re-
ported in the circulation of individuals who use marijuana,
but similar to other ex vivo studies of THC [17–20],
perhaps reflecting serum reduction of bioactivity by THC
in tissue culture models [21].
To explore the mechanism by which THC enhanced
LPS-stimulated TF expression and activity, selected signal
transduction events were investigated. Although TF expres-
sion is dependent on ERK1/2 and NF-κB signaling [2, 3],
phosphorylation of ERK1/2 and p65 or degradation of IκBα
were unchanged (Fig. 3a-d). Since signal transduction is un-
altered, it is unlikely that THC ubiquitously promotes in-
flammation, rather THC imparts an effect that is TF
specific. Moreover, TNFαor IL-8 mRNA and secreted pro-
tein increased modestly with THC treatment, while IL-6
was unchanged (Fig. 4). In addition, while THC treatment
results in modest increases in IL-8 and TNFα,THCmedi-
ated elevations in TF are of a greater magnitude. However,
as TNFαstimulates TF expression [22], small elevations in
TNFαexpression may act synergistically with other mecha-
nisms to further enhance THC mediated elevations in TF
expression and activity.
Our results are consistent with a mechanism of THC me-
diated elevation of TF expression at a post-transcriptional
level by inducing stabilization or preventing degradation of
TF mRNA. Recently, Poly(ADP-ribose)-polymerase(PARP)-
14 and tristetraprolin (TTP) were shown to cooperate to
mediate TF mRNA degradation [5]. While TTP regulates
mRNA transcripts of inflammatory mediators, such as
TNFα[23], the addition of PARP-14 renders the complex
TF specific. Since the magnitude and kinetics of TF mRNA
expression (Fig. 1e) differ from TNFα(Fig.4b),ourdata
suggest that TTP likely plays less of a role in THC medi-
ated elevations of TF compared to PARP-14.
Recreational marijuana use is prevalent, including
among individuals with IBD and HIV-1 infection [24, 25],
who are also at increased risk for coagulation disorders
[8,10].Inaddition,marijuanausersarelikelytocon-
sume alcohol [25]. Both acute binge drinking and
chronic alcohol use increase microbial translocation
and circulating endotoxin [26, 27]. Taken together with
our results, marijuana use alone or coupled with exces-
sive alcohol use, may also enhance circulating pro-
coagulant capacity.
mRNA
2 3 6 12
0.0
0.5
1.0
1.5
2.0
IL-6
Relative to control
24
1 2 3 6 12
0.0
0.5
1.0
1.5
2.0
TNF
Relative to control
A
B
Protein
24
24
1 2 3 6 12
0
1
2
3
IL-8
Relative to control
C
Time of LPS (hrs)
Fig. 4 Effect of THC on pro-inflammatory cytokine stimulated by
LPS. mRNA or cell supernatants (protein) from monocytes from at
least 4 donors were treated with vehicle or 30 μM THC for 30 min
prior to the addition of 100 ng/mL LPS for 24 h. Real-time quantitative
PCR or ELISAs for (a)IL-6,bTNFα,andcIL-8 were performed and
graphed relative to vehicle control at each time point (dotted line at 1).
Left panels show mean and standard error of donors
Williams et al. Journal of Inflammation (2015) 12:39 Page 5 of 6

Findings indicate that marijuana use may increase the
procoagulant potential of circulating monocytes and under-
score the importance of investigating the effects of
marijuana use in vivo. Recently, several cases of sudden
death in otherwise healthy individuals have linked acute
marijuana use to cardiovascular complications [28]. As use
of marijuana for both medicinal purposes and recreational
purposes increases, investigation and close monitoring of
coagulation related disorders is crucial, especially in individ-
uals with diseases characterized by microbial translocation
and dysregulated systemic inflammation.
Abbreviations
DMEM: Dulbecco’s Modified Eagle Medium; GAPDH: Gyceraldehyde
3-phosphate dehydrogenase; IBD: Inflammatory bowel disease;
IL-6: Interleukin-6; IL-8: Interleukin-8; LPS: Lipopolysaccharide; MAPK: Mitogen
activated protein kinase; NF-κB: Nuclear factor κB; PVDF: Polyvinyl difluoride;
SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
TF: Tissue factor; THC: Δ
9−
tetrahydrocannabinol; TNFα: Tumor necrosis factor
alpha.
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
JCW conceived of, designed, and executed experiments as well as wrote the
manuscript. TWK and JWS provided critical expertise and reviewed the
manuscript. BAG provided critical reagents and expertise and reviewed the
manuscript. NM provided essential expertise, critical analysis of results and
reviewed the manuscript. MMG provided critical analysis of results and
participated in writing of the manuscript. All authors read and approved the
final manuscript.
Acknowledgments
This work was supported in part by funding from the National Institute on
Drug Abuse (DA031017) and the University of Florida, department of
Pathology, Immunology, and Laboratory medicine Experimental Pathology
Innovative Grant awarded to J.C.W. J.C.W. is supported by the Laura McClamma
Fellowship at the University of Florida. Further support is provided by the
Stephany W. Holloway Endowed University Chair for AIDS Research (University
of Florida), University of Florida Cancer Center, and University of Florida Center
for Research in Pediatric Immune Deficiency. We thank Phillip Lichlyter and
Ashley Donnelly for technical support and acknowledge the University of
Florida Interdisciplinary Center for Biotechnology Research genomics core
facility for access to the ABI 7500 FAST instrument for Real Time PCR analysis
and the cellomics facility for access to LSR II flow cytometer.
Author details
1
Department of Pathology, Immunology and Laboratory Medicine, College of
Medicine, University of Florida, 2033 Mowry Road, Gainesville, FL 32610-3663,
USA.
2
Department of Molecular Medicine, Morsani College of Medicine,
University of South Florida, Tampa, FL, USA.
3
Department of Pediatrics,
Division of Allergy, Immunology and Rheumatology, School of Medicine,
Duke University, Durham, NC, USA.
4
Division of Hematology and Oncology,
Department of Medicine, McAlister Heart Institute, University of North
Carolina, Chapel Hill, NC, USA.
Received: 5 March 2015 Accepted: 4 June 2015
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