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Human Vγ2Vδ2 T Cells Augment
Migration-Inhibitory Factor Secretion and
Counteract the Inhibitory Effect of
Glucocorticoids on IL-1 β and TNF-α
Production
Lisheng Wang, Hiranmoy Das, Arati Kamath, Lin Li and
Jack F. Bukowski
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2002 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2002; 168:4889-4896; ;
doi: 10.4049/jimmunol.168.10.4889
http://www.jimmunol.org/content/168/10/4889
The Journal of Immunology
Human V␥2V␦2 T Cells Augment Migration-Inhibitory Factor
Secretion and Counteract the Inhibitory Effect of
Glucocorticoids on IL-1␤ and TNF-␣ Production1
Lisheng Wang, Hiranmoy Das, Arati Kamath, Lin Li, and Jack F. Bukowski2
T
he host response to inflammation and infection involves
the production of cytokines. Three pleiotropic cytokines,
macrophage migration-inhibitory factor (MIF),3 IL-1␤,
and TNF-␣, have broad biological effects. MIF is a pituitary peptide released during the physiological stress response, a proinflammatory macrophage cytokine secreted after LPS stimulation, and a
T cell product expressed as part of the Ag-dependent activation
response (1, 2). MIF counteracts the inhibitory effects of glucocorticoids on TNF-␣, IL-1␤, IL-6, and IL-8 production by monocytes
in response to stimulation with LPS in vitro (1). MIF is also involved in broad-spectrum pathophysiological reactions as an inflammatory cytokine. In mouse models, treatment with anti-MIF
Ab reduces mortality of septic shock (3), suppresses endotoxininduced fatal hepatic failure (4), inhibits rheumatoid arthritis (5),
and inhibits tumor growth (6, 7). MIF also augments resistance to
microbial infection (8), and shows endocrine and enzymatic functions (9, 10).
TNF-␣ and IL-1␤ are multifunctional cytokines and play pivotal
roles in inflammation and infection. For example, they are considered to be master cytokines in the pathobiology of septicemia and
septic shock (11, 12), and in chronic, destructive arthritis (13).
Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy,
Department of Medicine, Brigham and Women’s Hospital and Harvard Medical
School, Boston, MA 02115
Received for publication September 6, 2001. Accepted for publication March 6, 2002.
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.
1
This research was supported by grants from the National Institutes of Health and the
Arthritis Foundation.
2
Address correspondence and reprint requests to Dr. Jack F. Bukowski, Lymphocyte
Biology Section, Division of Rheumatology, Immunology, and Allergy, Department
of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Smith
Building, Room 526D, One Jimmy Fund Way, Boston, MA 02115. E-mail address:
[email protected]
3
Abbreviations used in this paper: MIF, migration-inhibitory factor; hu-SCID, SCID
mice reconstituted with human PBMC; IBA, isobutylamine; LB, Luria-Bertani;
TLR, Toll-like receptor.
Copyright © 2002 by The American Association of Immunologists
TNF-␣ and IL-1␤ may contribute to pathogenesis of thyroid autoimmunity (14 –16). They may also mediate host defense responses to neuroinflammation and cell death in neurodegenerative
conditions, in particular, multiple sclerosis and Parkinson’s and
Alzheimer’s diseases (17–22). In addition, TNF-␣ and IL-1␤ have
potent effects in the CNS, resulting in fever, induction of sickness
behavior, and activation of the hypothalamic-pituitary-adrenal axis
(reviewed in Refs. 23 and 24).
Release of cytokines can be harmful and sometimes lethal to the
host (25–29). Therefore, cytokine production must be reciprocally
and finely tuned in vivo, and imbalance of tuning could cause
immune system disorders. Glucocorticoids are one of the most
potent regulators of cytokine production (30, 31). Glucocorticoids
reduce the number of monocytes; lyse immature T cells; block
phospholipase A2 activity; down-regulate the synthesis and secretion of IL-1, IL-6, and TNF-␣ from activated monocytes and macrophages; and inhibit cytokine-induced transcription factors, such
as NF-␬B and AP1 (32, 33). The crucial role of glucocorticoids has
been demonstrated in a number of studies in which following adrenalectomy, challenge with TNF-␣ or IL-1 at doses that would be
well tolerated in adrenal-intact animals proves fatal (34). These
lethal effects can be prevented by steroid treatment (34).
␥␦ T cells comprise only 2–5% of CD3⫹ cells in human peripheral blood. Approximately 70% of these ␥␦ T cells coexpress
V␥2 and V␦2 TCR chains, and are uniformly reactive to nonpeptide organophosphate and alkylamine Ag, without CD1 or MHC
restriction (35, 36). Several lines of evidence suggest that these
cells play an important role in immunoregulation (37– 43). ␥␦ T
cells prime macrophages to produce TNF-␣ in response to LPS
stimulation (43), and augment production of macrophage-derived
NO (44). Depletion of ␥␦ T cells results in a decrease of TNF-␣,
IL-1␤, IFN-␥, and IL-6 gene expression in the spinal cord of mice
with autoimmune encephalomyelitis (45). However, the roles of
␥␦ T cells in the immunoregulation of cytokine production are still
poorly understood. It is unknown whether human ␥␦ T cells participate in regulating MIF secretion, and how ␥␦ T cells, glucocorticoids, and cytokines converge to give a unified physiological
0022-1767/02/$02.00
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In immune cells, proinflammatory cytokine gene expression is regulated by glucocorticoids, whereas migration-inhibitory factor
(MIF), a pleiotropic cytokine, has the unique property of counteracting the inhibitory effect of glucocorticoids on TNF-␣ and
IL-1␤ secretion. A few lines of evidence suggest that ␥␦ T cells play an important role in immunoregulation. However, it is
unknown whether human ␥␦ T cells participate in regulating MIF secretion, and how ␥␦ T cells, glucocorticoids, and cytokines
converge to give a unified physiological response. In this study, we demonstrate that human V␥2V␦2 T cells augment MIF
secretion. Remarkably, these V␥2V␦2 T cells, functioning similarly to MIF in part, counteracted inhibition of dexamethasone on
production of IL-1␤ and TNF-␣. SCID mice reconstituted with human PBMC that were mock depleted of V␦2 T cells and
repeatedly infected with lethal dose of Escherichia coli had shorter survival time than those reconstituted with PBMC that were
depleted of V␦2 T cells. Thus, human V␥2V␦2 T cells are likely to play broad-spectrum roles in immunoregulation and immunopathology by influencing MIF secretion and the immunomodulatory function of glucocorticoids. The Journal of Immunology,
2002, 168: 4889 – 4896.
4890
V␥2V␦2 T CELLS COUNTERACT INHIBITORY EFFECTS OF GLUCOCORTICOIDS
response. In this study, we demonstrate that V␥2V␦2 T cells augment MIF secretion and counteract the inhibitory effect of glucocorticoids on production of IL-1␤ and TNF-␣, suggesting that
V␥2V␦2 T cells are involved in immunoregulation. In an in vivo
hu-SCID model (SCID mice reconstituted with human PBMC),
human V␥2V␦2 T cells were found to participate in the pathogenesis of septic shock.
Materials and Methods
at the indicated time points were collected for analysis of MIF, IL-1␤, or
TNF-␣ by ELISA.
Detection of human IL-1␤, TNF-␣, and MIF in the tissue
culture supernatant
Human IL-1␤, TNF-␣, and MIF ELISA were performed according to the
procedures recommended by the manufacturers. The detection limit of the
assay was 15.6 pg/ml for IL-1␤, 7.8 pg/ml for TNF-␣, and 60 pg/ml
for MIF.
Intracellular cytokine staining
mAb ascites against T cell Ags used were as follows: control mAb (P3),
pan-␥␦ TCR (anti-TCR␦1), V␦1 (A13), V␦1/J␦1 (␦TCS1), V␦2 (BB3),
V␥2 (7A5), and CD3 (OKT3). The specificity of these mAbs is reviewed
in Porcelli et al. (46). Other reagents were purchased as follows: FITCconjugated F(ab⬘)2 goat anti-mouse IgM and IgG (catalog number
AMI4708; BioSource International, Camarillo, CA); isobutylamine (IBA;
catalog number I-3634; Sigma-Aldrich, St. Louis, MO); pamidronate (Novartis, East Hanover, NJ); PE-conjugated mouse anti-human TNF-␣ (catalog number 18630D; BD PharMingen, San Diego, CA), and anti-human
IL-1␤ (catalog number IC201P; R&D Systems, Minneapolis, MN); ELISA
human TNF-␣ set (catolog number 555212; BD PharMingen), and IL-1␤
set (catolog number 2687KI; BD PharMingen); anti-human MIF mAb (catalog number MAB289; R&D Systems); biotinylated anti-human MIF Ab
(catalog number BAF289; R&D Systems); human rMIF (catalog number
289-MF; R&D Systems); LPS (catalog 201, from Escherichia coli 0111:
B4; List Biological Laboratories, Campbell, CA); and Limulus amebocyte
lysate (catalog number GS003; Associates of Cape Cod, Falmouth, MA).
Human PBMC were cultured in RPMI medium with inclusion or exclusion
of 10 nM dexamethasone in the absence of LPS (Fig. 2), or in the presence
of LPS (Fig. 4). Four hours before staining with cellular surface marker,
monensin (GolgiStop; BD PharMingen) that enhanced intracellular cytokine accumulation was added in the medium. Cells were washed with PBS
and stained with surface marker AlexaFluor-conjugated 488 IgG control
Ab, pan-TCR␦1 (Abs purified and conjugated by our laboratory), or FITCconjugated anti-CD14. After two washes, cells were fixed with 2% formaldehyde in PBS and permeabilized with 0.5% (w/v) saponin (BD PharMingen). Intracellular TNF-␣ and IL-1␤ were stained with PE-conjugated Ab
in saponin buffer. After two washes, cells were resuspended in PBS and
analyzed using FACS flow cytometer (BD Biosciences, Mountain View,
CA) and Flowjo software (Tree Star, San Carlos, CA). V␦2 T cells themselves did not produce intracellular IL-1␤, TNF-␣, and IFN-␥ after 16 to
24 h culture in the medium containing 10% FBS or 1 ␮g/ml LPS (47) (L.
Wang, unpublished observation).
PBMC
Homozygous C.B-Igh-1b/Gbms-Prkdc(SCID)-Lyst(beige)N7 (SCID) male
mice, 5– 6 wk old, were purchased from Taconic Farms (Germantown, NY)
and maintained in microisolator cages. Animals were fed autoclaved food
and water, and all manipulations were performed under laminar flow. The
mice were weighed and randomly distributed into groups of 5–14 animals
with equal mean body weight. E. coli (ATCC 25922) was grown in LuriaBertani (LB) broth at 37°C until the culture reached early stationary phase.
E. coli was aliquoted (1 ml/vial) and stored in LB broth containing 10%
glycerol at ⫺80°C until use. Before infection, E. coli were washed once
with 30 ml PBS and plated on LB agar to determine CFU. The SCID mice
were injected i.p. with 0.5 ml RPMI medium containing 3 ⫻ 107 human
PBMC and 0.5 ml PBS containing 1–5 ⫻ 107 E. coli under aseptic conditions. For septic shock model, the mice were inoculated with E. coli
twice, 1 ⫻ 107 as first dose and 5 ⫻ 107 as second dose. The interval
between two bacterial infections was 20 h. The animals were observed four
times per day for their survival time.
Human PBMC obtained from random healthy donor leukopacks (DanaFarber Cancer Institute, Boston, MA) were isolated by Ficoll-Hypaque
centrifugation (Pharmacia, Piscataway, NJ). PBMC were cryopreserved in
FBS containing 10% DMSO at ⫺196°C until use.
Depletion of V␥2V␦2 T cells
Depletion of V␥2V␦2 T cells was performed by use of mouse anti-human
V␦2 Ab (BB3), or P3, an isotype-matched mock control, and goat antimouse IgG Dynabeads M-450 (catalog number 110.06; Dynal Biotech,
Oslo, Norway), according to the manufacturer’s instructions. For most depletions, P3, an isotype-matched control mAb, was substituted for the antiV␦2 mAb. For some depletions, anti-human V␦1 Ab (A13) was taken as an
alternative control and substituted for the anti-V␦2 mAb. V␦2 T cells constituted 70 –90% of total ␥␦ T cells for the PBMC used in our experiments.
Over 95% of V␦2 T cells, confirmed by surface marker staining and analysis of flow cytometry, were depleted. Since V␦2 T cell constituted only
about 2% of CD3⫹ cells in our experiments, depletion of V␦2 T cells from
PBMC with a specific V␦2 T cell Ab (BB3) did not show significant influence on percentages of CD14⫹ and other cells when analyzed by flow
cytometer (data not shown). We screened several donors by two-color fluorescence and found that 100% of V␦2-bearing T cells also expressed V␥2.
Although the V␦2 TCR chain paired with V␥1 or V␥3 has been described,
they are extremely rare, and there is no evidence that they respond to
nonpeptide Ags. Thus, the likelihood that we are studying a population
other than V␥2V␦2⫹ T cells is very remote.
Detection of endotoxin
Endotoxin levels in the Abs and reagents were assessed with the Limulus
amebocyte lysate kit according to the procedures recommended by manufacturer. The detection sensitivity of the assay was 0.03 EU/ml. Endotoxin
levels in P3 (isotype-matched mock control Ab) and BB3 (V␦2 Ab) Abs
were all negative at the concentration used for depletion of V␦2 T cells.
Stimulation of PBMC with dead bacteria and LPS in vitro
To determine whether ␥␦ T cells, under a physiological condition, play a
role in regulating monocytes to produce IL-1␤ in presence of dexamethasone, neither LPS nor dead E. coli was applied in the experiments (Fig. 2),
whereas, to determine whether ␥␦ T cells regulate PBMC to produce and
secrete IL-1␤ and TNF-␣ in a pathological circumstance, LPS or dead E.
coli was used in the experiments (Figs. 3 and 4), as described below.
PBMC were washed twice after 16-h preincubation with 1 mM IBA or
medium in presence or absence of dexamethasone. LPS (final concentration: 1 ␮g/ml) or dead E. coli (inactivated at 56°C for 2 h, final concentration: 5 ⫻ 105 CFU/ml) was added to each well. The culture supernatants
Engraftment and infection of SCID mice
Statistics
Values were expressed as means ⫾ SEM of the respective test or control
group. Statistical significance between control and test groups was calculated by Student’s t test (two-tailed) and among groups by analysis of
variations. Survival time was analyzed by Kaplan-Meier. Data were representative of two to four experiments.
Results
Human V␥2V␦2 T cells up-regulate MIF secretion
MIF plays a critical role in the systemic inflammatory response by
counter-regulating the inhibitory effect of circulating glucocorticoids on immune cell activation and proinflammatory cytokine
production (1). To determine whether V␥2V␦2 T cells regulate
MIF secretion, human PBMC were depleted or mock depleted of
V␦2 T cells and subsequently stimulated with LPS. The culture
supernatant was analyzed for MIF titer by ELISA. PBMC mock
depleted of V␦2 T cells secreted up to 2-fold more MIF than those
depleted of V␦2 T cells either in the absence (Fig. 1a) or presence
(Fig. 1b) of dexamethasone, suggesting that optimal MIF secretion
is dependent on presence of V␥2V␦2 T cells. Addition of 10 nM
dexamethasone to the culture, in agreement with others (1, 2),
inhibited MIF secretion (Fig. 1b). As an additional control, PBMC
depleted of V␦1 T cells had greater levels of MIF secretion compared with V␦2-depleted PBMC, after exposure to LPS (data not
shown). Optimum augmentation of MIF secretion by V␥2V␦2 T
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Ab and Ag reagents
The Journal of Immunology
cells occurred at 3– 6 h after stimulation, with a major loss of titer
by 9 h after stimulation (Fig. 1c).
Human V␥2V␦2 T cells counteract the inhibitory effect of
dexamethasone on IL-1␤ production
Since V␥2V␦2 T cells augmented the release of MIF (Fig. 1), we
speculated that these cells might also influence production and
secretion of IL-1␤. As monocytes are a major source of IL-1␤, we
first analyzed intracellular IL-1␤ production of monocytes at a
physiologic concentration of dexamethasone in the absence of LPS
stimulation, by use of two-color flow cytometry. In absence of
dexamethasone and LPS, 41.3 and 42.6% of monocytes produced
IL-1␤ in the absence or presence of V␥2V␦2 T cells, respectively.
In contrast, when PBMC were cultivated in 10 nM dexamethasone
(equivalent to a physiological level of bioactive cortisol in normal
human blood), monocytes reduced their intracellular IL-1␤ production by up to 30-fold in the absence of V␦2 T cells (Fig. 2).
This low level of intracellular IL-1␤ staining of monocytes in the
absence of V␦2 T cells was not due to cytokine release to the
extracellular medium, since there was no detectable IL-1␤ in the
culture supernatant in the absence of LPS or dead E. coli stimulation (Fig. 3a). These data suggest that V␥2V␦2 T cells counteract
the inhibitory effect of glucocorticoids on IL-1␤ production by
monocytes under physiologic conditions, resulting in an increased
storage pool of intracellular IL-1␤. If PBMC were stimulated with
LPS for 1– 6 h, ⬎90% of CD14⫹ monocytes produced intracellular
IL-1␤ and TNF-␣ in the absence of dexamethasone, and 30 – 60%
in the presence of dexamethasone, dependent on the concentration
of dexamethasone added. As a result, any potential difference in
intracellular cytokine production in the presence or absence of ␥␦
T cells was veiled by this overwhelming cytokine production during short-term (1– 6 h) exposure to LPS.
Since intracellular cytokine staining reflects a snapshot of accumulated cytokine by each individual cell, and only secreted cytokines affect the immunologic response, we then assessed IL-1␤
levels in the supernatant of PBMC in the presence of 10 nM dexamethasone. In the absence of stimulation with LPS or dead bacteria, PBMC secreted only trace amounts of IL-1␤, whereas, after
stimulation with LPS or dead bacteria, PBMC that were mock
depleted of V␦2 T cells secreted up to 2-fold more IL-1␤ than
PBMC depleted of V␦2 T cells (Fig. 3a). If the cells were cultivated in the medium containing a natural V␥2V␦2 T cell-specific
Ag, IBA (48, 49), and then stimulated with LPS, PBMC that were
mock depleted of V␦2 T cells secreted up to 4-fold more IL-1␤
than PBMC depleted of V␦2 T cells (Fig. 3a). Replacement of LPS
with dead E. coli (Fig. 3a), or replacement of IBA with the pharmaceutical V␥2V␦2 T cell-specific Ag, pamidronate (data not
shown), yielded similar results. These data suggest that V␥2V␦2 T
cell-specific Ags activate ␥␦ T cells, which then augment the ability of PBMC to secrete IL-1␤ upon exposure to LPS or dead
bacteria.
To determine whether MIF could reverse the inhibition of glucocorticoids on IL-1␤ secretion, we added 1 ng MIF to the cultures
containing 10 nM dexamethasone and 1 ␮g LPS. In this combination, PBMC depleted of V␦2 T cells resumed their IL-1␤ secretion nearly to the levels of PBMC that were mock depleted of V␦2
T cells (Fig. 3b). Whereas addition of exogenous MIF did not
significantly augment IL-1␤ secretion from the PBMC that were
mock depleted of V␦2 T cells, addition of 1 ng/ml MIF restored
IL-1␤ secretion from V␦2 T cell-depleted cultures to the levels
similar to those achieved in the presence of V␥2V␦2 T cells.
Human V␥2V␦2 T cells counteract the inhibition by
dexamethasone of TNF-␣ production and secretion
Since V␥2V␦2 T cells counteracted the inhibitory effect of glucocorticoids on IL-1␤ production (Fig. 2), we speculated that these
␥␦ T cells might also influence production and secretion of TNF-␣.
As monocytes are a major source of TNF-␣, we first analyzed the
effect of 10 nM dexamethasone on the intracellular TNF-␣ production of monocytes. In the absence of V␦2 T cells, only 0.045%
of CD14⫹ cells produced intracellular TNF-␣, whereas in presence
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FIGURE 1. Human V␥2V␦2 T cells augmented MIF secretion by
PBMC. Human PBMC were depleted or mock depleted of V␦2 T cells,
cultivated for 16 h, and then stimulated with 1 ␮g/ml LPS. The MIF levels
in the culture supernatant were determined by ELISA. PBMC mock depleted of V␦2 T cells secreted up to 2-fold more MIF than PBMC depleted
of V␦2 T cells after stimulation with LPS for 7 h (a). MIF secretion in
response to LPS stimulation was inhibited by addition of 10 nM dexamethasone (b). Time course of MIF secretion in absence of dexamethasone was
depicted (c). The levels of MIF before LPS stimulation were 270 –300
pg/ml in both depletion and mock depletion groups. The data of three
figures were produced from different individual PBMCs. Data were representative of two experiments.
4891
4892
V␥2V␦2 T CELLS COUNTERACT INHIBITORY EFFECTS OF GLUCOCORTICOIDS
of V␦2 T cells, 1.27% of CD14⫹ cells produced intracellular
TNF-␣ (Fig. 2). We then determined the TNF-␣ production of
monocytes in response to stimulation with LPS or heat-killed E.
coli. Similar to the results obtained with IL-1␤ (data not shown),
monocytes produced up to 2-fold more intracellular TNF-␣ in the
presence than in the absence of V␥2V␦2 T cells after 18-h exposure to LPS or heat-killed E. coli (Fig. 4a). These data suggest that
human V␥2V␦2 T cells, similar to mouse ␥␦ T cells (43), upregulate intracellular TNF-␣ production by human monocytes in
response to stimulation with LPS or dead bacteria.
We further assessed TNF-␣ secretion from PBMC in response
to stimulation with LPS. Human PBMC depleted of V␦2 T cells
secreted up to 2-fold less TNF-␣ as compared with PBMC that
were mock depleted of V␦2 T cells, when stimulated with LPS
(Fig. 4b). Since V␥2V␦2 T cells themselves did not produce and
secrete TNF-␣ in response to stimulation with LPS (47), these data
suggest that optimal TNF-␣ secretion by PBMC is dependent on
presence of V␥2V␦2 T cells.
To determine whether MIF could reverse the inhibition of glucocorticoids on TNF-␣ secretion, we added 1 ng MIF to the cultures containing 1 ␮M dexamethasone and 1 ␮g LPS. In this combination, PBMC depleted of V␦2 T cells resumed their TNF-␣
secretion nearly to the levels of PBMC that were mock depleted of
V␦2 T cells (Fig. 4c). Whereas addition of exogenous MIF did not
significantly augment TNF-␣ secretion from the PBMC that were
mock depleted of V␦2 T cells, addition of 1 ng/ml MIF restored
TNF-␣ secretion from V␦2 T cell-depleted cultures to levels similar to those achieved in the presence of V␥2V␦2 T cells.
Taken together, the above data suggest that V␥2V␦2 T cells
augment MIF secretion, counteract the inhibitory effect of glucocorticoids on production and secretion of IL-1␤ and TNF-␣, and
influence the interaction of MIF and glucocorticoids on cytokine
secretion by PBMC.
V␥2V␦2 T cells play a role in septic shock in vivo
Since MIF, TNF-␣, and IL-1␤ have been shown to be critical
mediators of septic shock (3, 11), we suspected that V␥2V␦2 T
cells might play a role in septic shock. To determine whether human V␥2V␦2 T cells play a role in bacterial infection in vivo, we
reconstituted SCID mice with human PBMC that were either mock
depleted or depleted of ␥␦ T cells, and challenged these mice with
E. coli. Six SCID mice i.p. challenged with 1 ⫻ 107 E. coli all died
of infection within 2 days postinfection. In contrast, five mice reconstituted with human PBMC and subsequently challenged with
the same dose of E. coli all survived, suggesting that human
PBMC play a crucial role against bacterial infection, and that residual mouse immune cells have negligible effects. This hu-SCID
model enables us to further investigate the roles of V␥2V␦2 T cells
in sepsis and septic shock. SCID mice were reconstituted with
human PBMC that were depleted or mock depleted of V␦2 T cells,
and subsequently infected with 1 ⫻ 107 CFU E. coli. After 20 h,
the mice received a second dose of E. coli (5 ⫻ 107 CFU). In
marked contrast to the experiments using low-dose bacterial infection (50), the mice receiving PBMC depleted of V␦2 T cells had
a longer survival time than those receiving PBMC that were mock
depleted of V␦2 T cells ( p ⫽ 0.0428, Fig. 5), suggesting that
V␥2V␦2 T cells play a role in septic shock.
Discussion
The immunoregulatory function of V␥2V␦2 T cells is still poorly
understood. In this study, we found that V␥2V␦2 T cells up-regulated MIF secretion (Fig. 1). Optimal production and secretion of
IL-1␤ and TNF-␣ were dependent on presence of V␥2V␦2 T cells
(Figs. 2, 3, and 4). Furthermore, V␥2V␦2 T cells counteracted the
inhibition by glucocorticoids of IL-1␤ and TNF-␣ production
(Figs. 2, 3b, and 4c).
Recent studies suggest that physiological levels of glucocorticoids are immunomodulatory rather than solely immunosuppressive, resulting in a shift of cytokine production from a Th1- to a
Th2-type pattern (30). Interruptions of this loop at any level, such
as genetic, surgery, infection, or pharmacological treatments, can
cause host susceptibility to infections and inflammatory diseases
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FIGURE 2. V␥2V␦2 T cells counteracted the inhibitory effects of glucocorticoids on intracellular IL-1␤ and TNF-␣ production by monocytes in the
absence of LPS stimulation. PBMC that were depleted or mock depleted of V␦2 T cells were cultivated in RPMI medium containing 10 nM dexamethasone
(equivalent to a physiological level of bioactive cortisol in normal human blood) for 16 h in the absence of LPS stimulation. Intracellular IL-1␤ and TNF-␣
production by monocytes was determined by flow cytometry using a two-color staining technique. Dexamethasone inhibited the ability of monocytes to
produce intracellular IL-1␤ and TNF-␣ by up to 30-fold in the absence of V␦2 T cells. Data were representative of three experiments. mIgG, Mouse IgG;
FSC-H, forward scatter height.
The Journal of Immunology
(30). We speculate that overactivation of glucocorticoid regulation, as occurred in absence of V␥2V␦2 T cells, might affect severity of infectious disease. Furthermore, in absence of V␥2V␦2 T
cells, MIF, TNF-␣, and IL-1␤, three pleiotropic and important cytokines for host resistance to bacterial infection (8, 51–57), had
reduced production or secretion. The relative overactivity of glucocorticoids and reduced levels of cytokine production and secretion might account for the severity of bacterial infection in vivo.
Consistent with this hypothesis, SCID mice reconstituted with human PBMC depleted of V␦2 T cells and subsequently infected
FIGURE 4. V␥2V␦2 T cells augmented TNF-␣ production and secretion in response to stimulation with LPS. a, Human PBMC that were depleted or mock depleted of V␦2 T cells were cultivated in RPMI medium
and stimulated with 1 ␮g/ml LPS for 18 h. Intracellular TNF-␣ production
by monocytes (stained with CD14 Ab) was determined by flow cytometry
using a two-color staining technique. Following a ubiquitous burst of
TNF-␣ production and secretion at the beginning of LPS stimulation,
monocytes produced up to 2-fold more intracellular TNF-␣ in the presence
of V␥2V␦2 T cells than in the absence of these cells 18 h after exposure to
LPS. b, Human PBMC depleted of V␦2 T cells secreted up to 2-fold less
TNF-␣ than PBMC that were mock depleted of V␦2 T cells when stimulated with 1 ␮g/ml LPS for 6 h. c, PBMC were cultivated in RPMI medium
containing 1 ng/ml MIF and 1 ␮g dexamethasone for 16 h and then stimulated with 1 ␮g/ml LPS for 6 h. MIF almost completely restored TNF-␣
secretion of V␦2 T cell-depleted cultures to the levels of PBMC cultures
that were mock depleted of V␦2 T cells. Data were representative of four
experiments. mIgG, Mouse IgG; FSC-H, forward scatter height.
with either E. coli, Morganella morganii, or Staphylococcus aureus had much higher mortality and bacterial load than those reconstituted with human PBMC that were mock depleted of V␦2 T
cells (50).
V␥2V␦2 T cells remarkably counteract the inhibitory effect of
glucocorticoids on IL-␤ production, and slightly on TNF-␣ production (Fig. 2). Interestingly, the increased IL-1␤ in presence of
V␥2V␦2 T cells was intracellularly stored in monocytes, but not
extracellularly secreted in the absence of LPS or bacterial stimulation. Once exposed to LPS or dead bacteria, monocytes began to
secrete IL-1␤ and TNF-␣, which was up-regulated by V␥2V␦2 T
cells (Figs. 3 and 4). The physiological significance of this finding
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FIGURE 3. V␥2V␦2 T cells augmented IL-1␤ secretion in response to
stimulation with LPS. a, PBMC depleted or mock depleted of V␦2 T cells
were cultivated in RPMI medium for 16 h in presence of 10 nM dexamethasone, then stimulated with either 10 ng/ml LPS or 5 ⫻ 105 heat-killed
E. coli for 3 h. IL-1␤ levels in the culture supernatant were determined by
ELISA. PBMC that were mock depleted of V␦2 T cells secreted up to
2-fold more IL-1␤ than PBMC depleted of V␦2 T cells. If PBMC were
cultivated in RPMI medium containing a natural V␥2V␦2 T cell-specific
Ag, IBA, for 16 h, and then stimulated with LPS or dead E. coli for 3 h,
PBMC mock depleted of V␦2 T cells secreted up to 4-fold more IL-1␤ than
PBMC depleted of V␦2 T cells. Replacing IBA with the pharmaceutical
V␥2V␦2 T cell-specific Ag, pamidronate (data not shown), produced similar results. Stimulation with 1 ␮g/ml LPS produced the results similar to
stimulation with 10 ng/ml LPS. b, PBMC were cultivated in RPMI medium
containing 10 nM dexamethasone and in the presence or absence of 1
ng/ml MIF for 16 h, and then stimulated with 1 ␮g/ml LPS for 6 h. Addition of 1 ng/ml MIF nearly restored the IL-1␤ secretion from PBMC that
had been depleted of V␦2 T cells, but not PBMC that had been mock
depleted of V␦2 T cells. Similar results were observed in the medium
containing 1 ␮M dexamethasone. Data were representative of three
experiments.
4893
4894
V␥2V␦2 T CELLS COUNTERACT INHIBITORY EFFECTS OF GLUCOCORTICOIDS
FIGURE 5. V␥2V␦2 T cells play a role in septic shock. SCID mice
were reconstituted with human PBMC that were depleted or mock depleted
of V␦2 T cells, and subsequently infected i.p. with 1 ⫻ 107 CFU E. coli.
After 20 h, the mice received a second dose of E. coli (5 ⫻ 107 CFU i.p.).
The mice receiving PBMC depleted of V␦2 T cells had a longer survival
time than those receiving PBMC that were mock depleted of V␦2 T cells
(p ⫽ 0.0428). Data were representative of two experiments.
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might be that, with assistance from V␥2V␦2 T cells, immune cells
accumulate a high level of intracellular cytokines ready to be secreted in case of infection, making the immune response earlier
and stronger. Since the prognosis of infection is dependent on the
speed of immune system reaction and pathogen proliferation, early
response to bacterial infection is crucial for the immune system to
eliminate pathogens. Therefore, in the absence of V␥2V␦2 T cells,
host resistance to bacterial infection would be impaired.
On the other hand, MIF, IL-␤, and TNF-␣ play important roles
in immunopathology. V␥2V␦2 T cells are capable of augmenting
MIF secretion and counteracting the inhibitory effect of glucocorticoids on production and secretion of IL-1␤ and TNF-␣, thereby
accounting for clinical symptoms. One piece of supportive evidence is that patients treated with the pharmaceutical V␥2V␦2 T
cell-specific Ag, pamidronate, had fever and influenza-like symptoms (58). This and other aminobisphosphonate drugs have been
widely used to inhibit osteoclastic bone resorption. As potent Ags,
aminobisphosphonates stimulate V␥2V␦2 T cells in a TCR-dependent, MHC- and CD1-unrestricted manner (48, 49). Influenza-like
symptoms occurred in these patients most likely due to the release
of cytokines, since TNF-␣ and IL-1␤ have been demonstrated to
have potent effects in the CNS, resulting in fever and sickness
behavior (23, 24). These cytokines, besides being directly produced from activated V␥2V␦2 T cells, were optimally generated
by other immune cells in a V␥2V␦2 T cell-dependent manner.
The interactions of V␥2V␦2 T cells and glucocorticoids implicate these T cells in a broad spectrum of immunoregulatory effects.
It is likely that V␥2V␦2 T cells participate in pathophysiological
reactions wherever glucocorticoids are involved, such as infection,
inflammatory, autoimmune, and allergic illnesses, including rheumatoid arthritis, systemic lupus erythematosus, Sjögren’s syndrome, allergic asthma, and atopic skin disease (30, 31, 59). Since
activated V␥2V␦2 T cells reduce the inhibitory effect of glucocorticoids on production and secretion of TNF-␣ and IL-1␤, any natural V␥2V␦2 T cell-specific Ags, such as nonpeptide alkylamine
(35), or organophosphate Ags (36), and drugs that activate
V␥2V␦2 T cells should be considered for their interference with
anti-inflammatory treatment regimens and disease outcomes.
Under extreme situations, overwhelming secretion of MIF,
TNF-␣, or IL-1␤ plays a critical role in the pathogenesis of septic
shock (3, 11, 12), resulting in tissue damage, multiple organ failure, and even death. Since V␥2V␦2 T cells augment MIF secretion
and counteract the inhibitory effect of glucocorticoids on production and secretion of TNF-␣ and IL-1␤, we suspect that these cells
might play a role in septic shock. To determine whether human ␥␦
T cells mediate antibacterial effects in vivo, we developed a huSCID infective model, which has proven to be a powerful method
for the study of human cells and tissues (60 – 64). We found that
SCID mice inoculated with either human PBMC or Salmonella
typhi all survived, whereas SCID mice receiving both PBMC and
S. typhi were all dead within 10 days postinfection (L. Wang,
unpublished observations), dependent on the dose of inoculated
human PBMC and bacteria. These data suggest that septic shock
resulted from interaction of human immune cells and bacteria. We
therefore reconstituted SCID mice with human PBMC that were
either mock depleted or depleted of ␥␦ T cells, then repeatedly
challenged these mice with lethal doses of E. coli. The mice receiving PBMC depleted of V␦2 T cells had a longer survival time
than those receiving PBMC mock depleted of V␦2 T cells (Fig. 5),
suggesting that human V␥2V␦2 T cells play a role in pathogenesis
of sepsis.
The mechanism by which V␥2V␦2 T cells counteract inhibition
of glucocorticoids on IL-1␤ and TNF-␣ production is unclear.
Monocytes constitutively secrete MIF, which could be augmented
by exposure to LPS (1, 2). T cells produce MIF as part of the
Ag-dependent activation response (1, 2). In our experiments, V␦2
T cells mediating immune regulation were observed at 16 h, but
not 6 h post-in vitro culture, suggesting that immune regulation of
these monocytes required considerable engagement time. In addition, we had experienced technical difficulties (e.g., a very poor
ratio of signal-noise) in distinguishing a small proportion of ␥␦ T
cells (about 2% of CD3⫹ cells in our experiments) from whole
PBMC in production of intracellular MIF. Therefore, we were unable to correlate MIF that may have been produced by a small
proportion of ␥␦ T cells at a certain time point, to an inhibitory
effect of glucocorticoids on IL-1␤ and TNF-␣ production. However, in the presence of ␥␦ T cells, MIF secretion by PBMC was
augmented (Fig. 1), and the inhibitory effect of glucocorticoids on
IL-1␤ and TNF-␣ production was counteracted (Figs. 2– 4).
We speculate that the major function of V␦2 T cells is to upregulate ␣␤ T cells and monocytes to produce more MIF, whereas
V␦2 T cell-produced MIF might constitute only a small proportion
of the MIF in the supernatant. In addition, since the use of antiMIF Ab could not significantly abrogate the enhanced cytokine
secretion seen in the presence of V␥2V␦2 T cells (data not shown),
it is possible that, besides augmentation of MIF secretion, V␥2V␦2
T cells counteract glucocorticoid activity by other unknown pathways. This possibility is supported by the fact that addition of
exogenous MIF only partially restored the deficit in IL-1␤ and
TNF-␣ production associated with ␥␦ T cell depletion (Figs. 3b
and 4c). This work demonstrates that ␥␦ T cells augment MIF
secretion and counteract the inhibitory effect of glucocorticoids on
IL-1␤ and TNF-␣ production. However, further studies are needed
to determine the exact mechanisms by which this counteraction
occurs.
␥␦ T cells did not produce IL-1␤, TNF-␣, and IFN-␥ in response
to the stimulation with LPS or dead E. coli (47). However, if these
␥␦ T cells were pretreated with IBA, a V␦2 T cell-specific Ag
secreted by live bacteria, then exposed to LPS, they started to
produce cytokines and to expand afterward (Kamath et al., manuscript in preparation). It is possible that human ␥␦ T cells, like
murine cells, express Toll-like receptors (TLRs) (65), or most
likely up-regulate TLR expression on monocytes, which are involved in LPS recognition. Recently, it was found that MIF regulated monocyte immune responses through modulation of TLR4
(66). Although we were unable to detect TLR2 and TLR4 on ␥␦ T
cells by flow cytometry, more sensitive methods, such as RT-PCR
used for detecting TLR expression on mouse ␥␦ T cells (65), or
microarray, might be alternative approaches for this purpose.
The Journal of Immunology
In conclusion, since V␥2V␦2 T cells augment MIF secretion and
counteract inhibition of glucocorticoids on production of IL-1␤
and TNF-␣, we speculate that these cells play more broad-spectrum roles in immunoregulation by influencing the glucocorticoid
immunomodulatory loop.
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