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2V
2 T Cells Augment Migration-Inhibitory Factor Secretion and Counteract the Inhibitory Effect of Glucocorticoids on IL-1
and TNF-
Production1
Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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 V2V2 T cells augment MIF secretion. Remarkably, these
V2V2 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 V2 T
cells and repeatedly infected with lethal dose of Escherichia
coli had shorter survival time than those reconstituted with
PBMC that were depleted of V2 T cells. Thus, human V2V2 T
cells are likely to play broad-spectrum roles in immunoregulation and
immunopathology by influencing MIF secretion and the immunomodulatory
function of glucocorticoids. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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 endotoxin-induced 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). TNF- and IL-1 may contribute to pathogenesis
of thyroid autoimmunity (14, 15, 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, 18, 19, 20, 21, 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, 26, 27, 28, 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 V2 and V2 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, 38, 39, 40, 41, 42, 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 response. In this
study, we demonstrate that V2V2 T cells augment MIF secretion and
counteract the inhibitory effect of glucocorticoids on production of
IL-1 and TNF-, suggesting that V2V2 T cells are involved in
immunoregulation. In an in vivo hu-SCID model (SCID mice
reconstituted with human PBMC), human V2V2 T cells were found to
participate in the pathogenesis of septic shock.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAb ascites against T cell Ags used were as follows: control mAb
(P3), pan- TCR (anti-TCR1), V1 (A13), V1/J1
(TCS1), V2 (BB3), V2 (7A5), and CD3 (OKT3). The specificity of
these mAbs is reviewed in Porcelli et al. (46). Other
reagents were purchased as follows: FITC-conjugated
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).
PBMC
Human PBMC obtained from random healthy donor leukopacks (Dana-Farber 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 V2V2 T cells
Depletion of V2V2 T cells was performed by use of mouse
anti-human V2 Ab (BB3), or P3, an isotype-matched mock control,
and goat anti-mouse 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 anti-V2 mAb. For some depletions,
anti-human V1 Ab (A13) was taken as an alternative control and
substituted for the anti-V2 mAb. V2 T cells constituted
70–90% of total T cells for the PBMC used in our experiments.
Over 95% of V2 T cells, confirmed by surface marker staining and
analysis of flow cytometry, were depleted. Since V2 T cell
constituted only about 2% of CD3+ cells in our
experiments, depletion of V2 T cells from PBMC with a specific V2
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 V2-bearing T cells also
expressed V2. Although the V2 TCR chain paired with V1 or
V3 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
V2V2+ 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 (V2 Ab) Abs were all negative at the concentration used for
depletion of V2 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
µg/ml) or dead E. coli (inactivated at 56°C for 2
h, final concentration: 5 x 105 CFU/ml) was
added to each well. The culture supernatants at the indicated time
points were collected for analysis of MIF, IL-1, or TNF- by
ELISA.
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, 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
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-TCR1 (Abs purified and conjugated by our
laboratory), or FITC-conjugated 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). V2 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 µg/ml LPS (47) (L. Wang,
unpublished observation).
Engraftment and infection of SCID mice
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 Luria-Bertani (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 x 107 human PBMC and 0.5 ml PBS containing 1–5 x 107 E. coli under aseptic conditions. For septic shock model, the mice were inoculated with E. coli twice, 1 x 107 as first dose and 5 x 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.
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2V2 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 V2V2 T cells regulate MIF
secretion, human PBMC were depleted or mock depleted of V2 T cells
and subsequently stimulated with LPS. The culture supernatant was
analyzed for MIF titer by ELISA. PBMC mock depleted of V2 T cells
secreted up to 2-fold more MIF than those depleted of V2 T cells
either in the absence (Fig. 1a) or presence (Fig. 1b) of dexamethasone, suggesting that optimal MIF secretion
is dependent on presence of V2V2 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 V1 T cells had greater levels
of MIF secretion compared with V2-depleted PBMC, after exposure to
LPS (data not shown). Optimum augmentation of MIF secretion by
V2V2 T cells occurred at 3–6 h after stimulation, with a major
loss of titer by 9 h after stimulation (Fig. 1c).
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2V2 T cells counteract the inhibitory effect of
dexamethasone on IL-1 production
Since V2V2 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 V2V2 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 V2 T cells (Fig. 2). This low level of intracellular
IL-1 staining of monocytes in the absence of V2 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
V2V2 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 V2 T
cells secreted up to 2-fold more IL-1 than PBMC depleted of V2 T
cells (Fig. 3a). If the cells were cultivated in the medium
containing a natural V2V2 T cell-specific Ag, IBA (48, 49), and then stimulated with LPS, PBMC that were mock depleted
of V2 T cells secreted up to 4-fold more IL-1 than PBMC depleted
of V2 T cells (Fig. 3a). Replacement of LPS with dead
E. coli (Fig. 3a), or replacement of IBA with the
pharmaceutical V2V2 T cell-specific Ag, pamidronate (data not
shown), yielded similar results. These data suggest that V2V2 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 µg LPS. In this combination,
PBMC depleted of V2 T cells resumed their IL-1 secretion nearly
to the levels of PBMC that were mock depleted of V2 T cells (Fig. 3b). Whereas addition of exogenous MIF did not significantly
augment IL-1 secretion from the PBMC that were mock depleted of
V2 T cells, addition of 1 ng/ml MIF restored IL-1 secretion from
V2 T cell-depleted cultures to the levels similar to those achieved
in the presence of V2V2 T cells.
Human V2V2 T cells counteract the inhibition by dexamethasone
of TNF- production and secretion
Since V2V2 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 V2 T cells, only 0.045%
of CD14+ cells produced intracellular TNF-,
whereas in presence of V2 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 V2V2 T cells after 18-h exposure to LPS or heat-killed
E. coli (Fig. 4a).
These data suggest that human V2V2 T cells, similar to mouse
T cells (43), up-regulate 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 V2 T cells secreted up
to 2-fold less TNF- as compared with PBMC that were mock depleted of
V2 T cells, when stimulated with LPS (Fig. 4b). Since
V2V2 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
V2V2 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 µM dexamethasone and 1 µg LPS. In this combination,
PBMC depleted of V2 T cells resumed their TNF- secretion nearly
to the levels of PBMC that were mock depleted of V2 T cells (Fig. 4c). Whereas addition of exogenous MIF did not significantly
augment TNF- secretion from the PBMC that were mock depleted of
V2 T cells, addition of 1 ng/ml MIF restored TNF- secretion from
V2 T cell-depleted cultures to levels similar to those achieved in
the presence of V2V2 T cells.
Taken together, the above data suggest that V2V2 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.
V2V2 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
V2V2 T cells might play a role in septic shock. To determine
whether human V2V2 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 x
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 V2V2 T cells in sepsis and septic shock.
SCID mice were reconstituted with human PBMC that were depleted or mock
depleted of V2 T cells, and subsequently infected with 1 x
107 CFU E. coli. After 20 h, the
mice received a second dose of E. coli (5 x
107 CFU). In marked contrast to the experiments
using low-dose bacterial infection (50), the mice
receiving PBMC depleted of V2 T cells had a longer survival time
than those receiving PBMC that were mock depleted of V2 T cells
(p = 0.0428, Fig. 5), suggesting that V2V2 T cells
play a role in septic shock.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2V2 T cells is still
poorly understood. In this study, we found that V2V2 T cells
up-regulated MIF secretion (Fig. 1). Optimal production and secretion
of IL-1 and TNF- were dependent on presence of V2V2 T cells
(Figs. 2, 3, and 4). Furthermore, V2V2 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
(30). We speculate that overactivation of glucocorticoid
regulation, as occurred in absence of V2V2 T cells, might affect
severity of infectious disease. Furthermore, in absence of V2V2 T
cells, MIF, TNF-, and IL-1, three pleiotropic and important
cytokines for host resistance to bacterial infection (8, 51, 52, 53, 54, 55, 56, 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 V2 T cells and
subsequently infected 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 V2 T cells (50).
V2V2 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 V2V2 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 V2V2 T
cells (Figs. 3 and 4). The physiological significance of this finding
might be that, with assistance from V2V2 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 V2V2 T cells, host resistance to
bacterial infection would be impaired.
On the other hand, MIF, IL-, and TNF- play important roles in
immunopathology. V2V2 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 V2V2 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 V2V2 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 V2V2 T cells, were optimally generated by other
immune cells in a V2V2 T cell-dependent manner.
The interactions of V2V2 T cells and glucocorticoids implicate
these T cells in a broad spectrum of immunoregulatory effects. It is
likely that V2V2 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 V2V2 T cells reduce the inhibitory effect of
glucocorticoids on production and secretion of TNF- and IL-1, any
natural V2V2 T cell-specific Ags, such as nonpeptide alkylamine
(35), or organophosphate Ags (36), and drugs
that activate V2V2 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 V2V2 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 hu-SCID infective
model, which has proven to be a powerful method for the study of human
cells and tissues (60, 61, 62, 63, 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 V2 T cells had a longer survival time than those
receiving PBMC mock depleted of V2 T cells (Fig. 5), suggesting that
human V2V2 T cells play a role in pathogenesis of sepsis.
The mechanism by which V2V2 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, V2 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 V2 T cells is to up-regulate
T cells and monocytes to produce more MIF, whereas V2 T
cell-produced MIF might constitute only a small proportion of the MIF
in the supernatant. In addition, since the use of anti-MIF Ab could
not significantly abrogate the enhanced cytokine secretion seen in the
presence of V2V2 T cells (data not shown), it is possible that,
besides augmentation of MIF secretion, V2V2 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 V2 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.
In conclusion, since V2V2 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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Footnotes |
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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: jbukowski{at}rics.bwh.harvard.edu
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.
Received for publication September 6, 2001. Accepted for publication March 6, 2002.
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  K.-J. Puan, C. Jin, H. Wang, G. Sarikonda, A. M. Raker, H. K. Lee, M. I. Samuelson, E. Marker-Hermann, L. Pasa-Tolic, E. Nieves, et al. Preferential recognition of a microbial metabolite by human V{gamma}2V{delta}2 T cells Int. Immunol., May 1, 2007; 19(5): 657 - 673. [Abstract] [Full Text] [PDF]
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