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Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) in the same cultures. This suggests that T cell activation in
hypoxic conditions in vivo may lead to different patterns of lymphokine
secretion and accumulation of cytokines (e.g., vascular endothelial
growth factor) affecting endothelial cells and vascular
permeabilization. Thus, although higher numbers of cells survive and
are activated during 20% oxygen incubation in vitro, the CTL which
develop at 2.5% oxygen are more lytic with higher levels of activation
markers. It is concluded that the ambient 20% oxygen tension (plus
2-ME) is remarkably well suited for immunologic specificity and
cytotoxicity studies, but oxygen dependence should be taken into
account during the design and interpretation of results of in vitro T
cell development assays and gene expression studies in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
HIF-2, which are required for activation of genes encoding
glucose transporters and glycolytic enzymes, as well as for synthesis
of such biologically active molecules as vascular endothelial growth
factor (VEGF) and inducible NO synthase (11, 12). In addition to changes in the patterns of gene expression and cell metabolism, low oxygen tension may affect the balance of SH and S-S groups implicated in maintenance of the properly folded state of proteins in the cytosol (13). Hypoxia may also cause changes in regulation of cellular processes due to the decrease in reactive oxygen species formation and action in many types of cells including T cells (14, 15).
Although secondary lymphoid organs are of utmost importance in immune cell development and functions, it was not known whether lymphocytes are exposed to hypoxic or normoxic environment in different compartments of thymus, spleen, and lymph nodes, and whether hypoxic exposure may affect immune response.
In this study, we have evaluated the effects of hypoxia on lymphocyte
differentiation and effector functions and addressed the following
questions. 1) What is the oxygen tension in different compartments of
murine lymphoid organs? 2) What are the effects of physiologically
relevant hypoxia on T cell differentiation vs effector functions of
differentiated T cells? 3) What is the effect of hypoxia on hypoxia
response element (HRE)-containing gene products (e.g., VEGF) vs
non-HRE-containing gene products (e.g., IL-2 and IFN-) in
TCR-activated lymphocytes?
The observations suggest that hypoxic exposure of T cells in vivo may significantly alter TCR-triggered lymphokine secretion patterns and CTL development without an appreciable effect on the lethal hit delivery by CTL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 and DBA/2 mice were maintained in pathogen-free National Institutes of Health animal facilities. Mice were 6–10 wk old, and four to eight animals were used in each experiment.
Oxygen measurements
Oxygen tension measurements in organs of DBA/2 mice were conducted as previously described (16) using a recessed-tip oxygen microelectrode with a tip diameter of 9–12 µm (17) and a micromanipulator (model M01202E; Narishige, Tokyo, Japan). Electrode current was measured with a microsensor (Chemical Microsensor model 201; Diamond General, Ann Arbor, MI) and recorded on a computer using CODAS software (AT-codas; Dataq Instruments, Akron, OH). Measurements were conducted in a Faraday cage which minimized extraneous electronic noise. The electrode was calibrated both before and after the measurements with four certified gas mixtures in saturated water. After each animal was sacrificed, the electrode remained in the organ to verify the null point of zero oxygen tension in the organ. At least three mice were used for each organ measured, while two to four tracks were taken sequentially of each organ.
Cells and medium
Splenocytes were isolated from adult spleens ex vivo and incubated in RPMI 1640 (Biofluids, Rockville, MD) supplemented with 5% dialyzed FCS (heat inactivated) and 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 1 mM HEPES, and nonessential amino acids (RP5). CTL clone BK1 (an anti-keyhole limpet hemocyanin (KLH) peptide), CD4+ CTL clone, and anti-H2d CTL clone GD1 (FasL deficient, derived from gld mice) were maintained according to published procedures (18, 19).
Unless otherwise indicated, medium for cultures conducted at 20% oxygen contained 50 µM 2-ME, while medium for cultures conducted at 1 or 2.5% oxygen concentrations did not. Culture vessels for MLCs were six-well plates filled with 9 ml of RP5 medium and a cell density of 2.5 million cells/ml medium (2.0 million effector and 0.5 million stimulators). Cells to be cultured at 1 or 2.5% oxygen were centrifuged and resuspended in culture medium saturated with a certified gas mixture containing 1 or 2.5% oxygen, 5.0% CO2, and 94 or 92.5% N2 (Robert’s Oxygen Company, Rockville, MD). Cells were incubated at various oxygen tensions in a NAPCO 7000 (NAPCO, Winchester, VA) three-gas (CO2, N2, and O2) incubator.
Cell culture and CTL assay
T cell development was studied during incubation of splenocytes isolated from C57BL/6 mice (responders) with irradiated (3,000 rads) splenocytes from DBA/2 mice stimulators at various oxygen tensions.
CTL cytotoxicity was measured in a routine 4-hr, 37°C, 51Cr-release assay and calculated as described earlier (20). The proportions of CD4+ and CD8+ cells in cultures were evaluated in parallel by flow cytometry.
Monoclonal Abs
FITC- or allophycocyanin-conjugated rat anti-mouse CD4
(clone RM-4-5), R-PE -conjugated rat anti-mouse CD25 (clone PC61),
FITC- or allophycocyanin-conjugated rat anti-mouse CD8a
(clone 53-67), PE-conjugated armenian hamster anti-mouse TCR
-chain (clone H57-597), PE-conjugated rat anti-mouse LFA-1
(CD11a, clone 2D7), PE- or allophycocyanin-conjugated rat
anti-mouse IL-2 (clone JES6-1A12), PE- or
allophycocyanin-conjugated rat anti-mouse IFN- (clone XMG1.2),
and anti-mouse CD3 mAb (clone 145-2C11) were purchased from BD
PharMingen (San Diego, CA).
Cytokine analysis
Intracellular staining of TCR-activated cells to evaluate
cytokine production in situ was done in permeabilized cells using
anti-IFN- and IL-2 mAb as described recently (21)
using 2 µM (final concentration) monensin (CalBiochem, La Jolla, CA)
and saponin buffer (PBS containing 0.1% (w/v) saponin, 0.1% BSA, 0.01
M HEPES, and 0.1% sodium azide) after 4 h of 2C11 anti-CD3
mAb restimulation. After staining with indicated anti-cytokine mAb,
cells were analyzed by flow cytometry.
Concentrations of IL-2 and IFN- in cell-free supernatants from MLC
were determined using ELISA kits (Amersham Pharmacia Biotech,
Piscataway, NJ). VEGF production was assayed using ELISA kits (R&D
Systems, Minneapolis, MN) according to the manufacturer’s
instructions.
Flow cytometry
Analysis of cell surface Ag expression and of cytokine expression in situ was performed as described earlier (21, 22). Flow cytometry data acquisition and analysis were done on a FACSCalibur using FACS research software and CellQuest programs (BD Biosciences, Mountain View, CA).
Statistics
Standard errors were determined using the Microsoft Excel spreadsheet program (Microsoft, Redmond, WA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Standard in vitro culture systems for CTL generation have a
microenvironment with a pH of 7.3, a CO2
concentration of 5% (v/v), and an oxygen tension of 
20%, with 50
µM 2-ME added to the culture. Measurements of pH in spleen tissue
yielded values of 7.20 and 7.21 in rat and cat, respectively (23, 24), and by extrapolating data on measurements of
CO2 in blood, it is accepted that the tissue
CO2 tension is 5% (25, 26). It
was important to evaluate the distribution of oxygen tension in
different compartments of lymphoid organs. Therefore, we proceeded to
directly measure oxygen tension in several lymphoid tissues, including
the spleen.
We observed low (between 4 and 34 torr, or 0.5–4.5%) oxygen tensions
in lymphoid organs (Fig. 1
) as compared
with 150 torr or 20% oxygen concentration in the atmosphere
(27). Fig. 1A shows oxygen level measurements
as the electrode is incrementally (50 µm per step) inserted into the
spleen. The oxygen tension is relatively higher near the splenic artery
(Fig. 1, B and C, tracks 3,
4, and 6) compared with the more distal locations
in the spleen (Fig. 1, B and C, tracks
5 and 7). Similarly low oxygen tensions were detected
in other lymphoid organs (data not shown).
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) or by incubating cells at the same
oxygen tension (20%) but in the presence or absence of reducing agent
2-ME (data not shown).
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Fas ligand- and perforin-mediated lethal hit delivery by differentiated CTL are not affected by an oxygen tension
Experiments taking advantage of genetic tools to discriminate
between possible differences in effects of changes in redox environment
on FasL/Fas- vs perforin-mediated cytotoxicity are described in Fig. 2
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Incubation of Fas-expressing cells at hypoxic conditions with
anti-Fas mAb established that the Fas-triggered cell death pathway
is not dependent on redox potential (data not shown); however, because
FasL-mediated cytotoxicity by CTL requires the TCR-triggered
up-regulation of Fas ligand (28), we directly tested
whether CTL FasL-mediated cytotoxicity is dependent on redox potential
(Fig. 2B). In these experiments, the redox dependence of
FasL-mediated cytotoxicity by CTL, which requires TCR-triggered
up-regulation of Fas ligand (28), was tested using a BK1
anti-KLH peptide, CD4+ CTL clone that
operates using only the FasL-mediated mechanism of lethal hit delivery
(Fig. 2B). A similar result was observed in studies of the
anti-Fas mAb/Fas-triggered cell death and in experiments using
FasL-deficient CTL clone GD1, which uses only perforin-mediated
cytotoxicity and kills equally well at 2.5 and 20% oxygen (Fig. 2A and data not shown). These experiments show that
FasL-mediated cell death is not affected by redox changes in the
environment.
Such insensitivity of both FasL- and perforin-mediated lethal hit delivery pathways to changes in redox potential is in stark contrast to the oxygen tension dependence of processes of CTL development.
Physiologically low oxygen tensions favor more lytic CD8+ CTL development
Routinely used assays of CTL development are performed at ambient oxygen tension (20% plus 2-ME), and this allows for accumulation of CTL after 5 or 6 days of culture in CO2 incubators. We confirmed that the addition of 2-ME (29, 30) was necessary for CTL development at ambient (20%) oxygen tension (data not shown), while the presence of 5 x 10-5 M 2-ME at lower oxygen tensions was not conducive for CTL development (1 or 3% oxygen, data not shown). It was important to compare the outcome of routinely used in vitro 20% oxygen with 50 µM 2-ME vs more physiologically relevant conditions of incubation of lymphocytes at 2.5% oxygen without 2-ME.
It is shown in Fig. 3A that at
day 5 there is no difference between cytolytic activity of CTL
developed at 2.5 vs 20% oxygen if calculations of numbers of effector
cells in E:T ratios were performed per total viable cells. However, it
was noticed that the CD4:CD8 ratio in these cultures was different
between hypoxic and normoxic conditions (Fig. 3B). Although
there were more CD8 than CD4 at 20% oxygen tensions (CD4:CD8
1:2), the opposite was observed at hypoxic conditions as the CD4/CD8
ratio was much higher (3:1) among MLC cells. This, taken together
with equal lytic activity of both total cell cultures (Fig. 3A), suggested that while there were fewer
CD8+ T cells developed at hypoxic conditions,
their lytic activity could be much higher per
CD8+ CTL basis. This prompted an investigation
where we tested both cytotoxicity and CD4 vs CD8 numbers of cells by
flow cytometry to enable the recalculation of cytotoxicity per
CD8+ CTL basis. The lytic potential of CTL in
these experiments was estimated by recalculating the results of the
51Cr release assay as the
CD8+ CTL:target cell ratio in addition to
routinely used numbers of total viable effector cells.
With these calculations, it was found that hypoxic conditions favor the
development of much more lytic CD8+ CTL (see Fig. 3C). Indeed, it is shown that CD8+ CTL
cells developed under hypoxic conditions possess a 7-fold higher
killing potential as measured by lytic units per million cells (Fig. 3C, inset). Thus, the much larger number of
less lytic CD8+ cells that accumulate at 20%
oxygen may misleadingly mask differences between the development of
lytic CTL under these two conditions at day 5 of MLC (Fig. 3A) if results are not compared as
CD8+ CTL: target ratio.
To differentiate between Ag-driven expansion of specific TCR-expressing
T cells and the possible expansion of bystander
CD8+ T cells, we used the OVA-specific
TCR-transgenic mice. Using this system we found, in agreement with our
MLC data in Figs. 3 and 4, that OT-1 CTLs
expanded more quickly under 20% oxygen than under 2.5% oxygen tension
when stimulated with the OT-1 specific peptide, SIINFEKL (Table I).
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, A and B). Furthermore, the numbers and
proportions of CD8+ T cells continued to increase
from day 5 to day 6 (Fig. 4B and data not shown) at hypoxic
2.5%. In contrast, it appears that the numbers and proportions of CD8
at day 6 at 20% are decreasing due to increased cell death (data not
shown). Even more striking differences were observed at day 6 of MLC in
regard to higher CD8+ CTL activity (Fig. 4C). When recalculated on per
CD8+ CTL basis a dramatic increase in lytic units
from 700 to 3000 was observed with CD8+ CTL at
hypoxic conditions, while at 20% CD8+ CTL remain
the same (Fig. 4C, inset).
The data above lead us to investigate both numbers and the activation
state of the CD8+ CTL cells. Table II shows that at least three times more
CD8+ cells are developed in MLC at 20% than at
2.5% oxygen. Not only are there more CD8+ cells,
but a 5-fold larger number of CD8+ cells also
express the IL-2R (CD25) at 20% oxygen. However, flow cytometry
analysis indicates that even though there are fewer
CD25+CD8+ T cells at 2.5%,
those CD8+ T cells that are activated at low
oxygen tension have a higher surface density of
CD25+ (mean fluorescence intensity = 807 at
day 5 at 2.5% vs 146 at 20% oxygen) (see Table II). Thus, although
more cells survive and are activated at 20% oxygen, cells at 2.5%
oxygen develop more slowly, have a higher level of activation, and are
much more lytic.
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To evaluate the effect of oxygen tension changes on cytokine
secretion and consumption, the levels of VEGF, IFN-, and IL-2
were determined in MLC culture supernatants by ELISA (Fig. 5, A and B) and on
the single-cell level in situ by flow cytometry (Fig. 6).
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It was shown with different types of cells, although not with T
lymphocytes, that the adaptation to low oxygen tension proceeds through
activities of HIF-1 and HIF-2, which trigger transcription of
HRE-containing genes (11). One of these genes is VEGF
(11), and we compared the effect of hypoxia on the
accumulation of VEGF and the non-HRE-containing IL-2, IFN- genes
(Fig. 5A) in activated lymphocytes.
The hypoxia-induced enhancement of cytokine accumulation was observed
in studies of VEGF during MLC at 2.5 vs 20% oxygen (Fig. 5B). No VEGF accumulation was found in supernatants of T
cells until the day 4 of incubation at 20% oxygen (Fig. 5B). In contrast, VEGF was detected in supernatants as soon
as the day 2 at hypoxic, 2.5% oxygen cultures. The normoxic, 20%
oxygen incubation did eventually result in accumulation of VEGF, but
the total amount of VEGF at hypoxic 2.5% was higher, suggesting that
there is a combined, additive effect of HRE-dependent and
TCR-activation-dependent VEGF production by T cells. The accumulation
of IL-2 in the 20% oxygen MLC supernatant at its peak was about 2-fold
higher than that found in the 2.5% MLC supernatant (Fig. 5C). Furthermore, the increase in the accumulation and the
decrease in IL-2 was also much greater in the 20% oxygen tension MLC
supernatant. Similarly, the rate of increase and the total amount of
IFN- in the 20% oxygen supernatant was much higher compared with
the 2.5% culture (Fig. 5D).
The experiment in Fig. 6 was designed to compare the production of IL-2
and IFN- at different oxygen tensions on a per cell basis. It is
shown that the rate of production, as measured by the intensity of
cytokine labeling by mAb, is about equal on all days for both cytokines
in both culture conditions. Furthermore, it appears that the percentage
of IL-2 secreting T cells is about the same on all days. However,
because the absolute number of TCR-activated, IL-2-producing cells is
much higher at 20% oxygen (as shown in Table II), the ELISA
measurements of IL-2 in supernatants yielded a higher concentration of
IL-2 at 20% than at 2.5% oxygen. In contrast, the proportion of
IFN--producing cells is much higher at 20% than at 2.5% oxygen. It
is shown in Fig. 6 that the proportion of IFN--producing CTLs is at
least 3-fold higher during days 3 and 4 of incubation.
Altogether, these experiments demonstrate that less VEGF, but much more
IFN- and IL-2 as well as much more IFN--producing
CD8+ and CD4+ T cells were
accumulated under normoxic conditions than at hypoxic conditions (Figs. 5 and 6). This may partially explain differences in cell numbers (Table II) as due to the IL-2-dependent growth and the proliferation advantage
of T cells at 20% oxygen.
High cell surface density of TCR/CD3 complex and cell adhesion LFA-1 molecules on CD8+ T cells at hypoxic conditions of MLC
Attempts to explain the increase in lytic activity of CTL from day
5 to day 6 at 2.5 vs 20% oxygen incubations (Figs. 3 and 4) included
evaluations of expression of recognition and cell adhesion molecules
(Fig. 7), of lytic molecules perforin and FasL, as well as comparison
of levels of CTLA-4 (data not shown). No differences between hypoxic
and normoxic conditions of incubations were found in levels of
expression of perforin, FasL, and regulatory CTLA-4 molecules as
determined by flow cytometry (data not shown). In contrast, levels of
expression of TCR and LFA-1 were higher on CD8+
cells at 2.5% oxygen tension, and much higher proportions of
CD8+ T cells could be classified as
TCRhighLFA-1high at 2.5%
oxygen than at 20% oxygen (Fig. 7).
Importantly, the greatest increase in proportion of
TCRhighLFA-1high
CD8+ T cells was observed at the time (days 5–6)
when the most lytic CD8+ CTL were detected at 2.5
vs 20% oxygen (see Figs. 3 and 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-2) in lymphocytes.
Indeed, the lethal hit delivery by CTL is not affected by hypoxia (Fig. 2). The observations of similar lethal hit delivery by CTL at 1, 2.5,
and 20% oxygen (Fig. 2 and data not shown) are conceptually appealing
and may reflect the fidelity of functions of this effector arm of
immune system in all tissue compartments. Otherwise, hypoxic areas,
such as areas of inflammation, would be providing "safe havens" for
infected cells. These data may also settle the longstanding controversy
regarding studies of the effect of oxygen tension on CTL lethal hit
delivery and processes of cell death (3, 32, 33, 34). It
appears that processes of cell death do require very low levels of
oxygen, but there are no differences in CTL-induced target cell death
when oxygen tension is varied between 1 and 20%.
In contrast to effector lytic functions of differentiated CTL, the
development of CTL and patterns of lymphokine accumulation were very
different at 2.5 vs 20% oxygen (
Figs. 3–5). An unexpected observation
was that low oxygen tension conditions result in development of
CD8+ CTL (Figs. 3 and 4), which were more lytic.
Studies of lymphokine accumulation in supernatants, in parallel with
estimations of IFN- and IL-2 on a per cell basis (Figs. 5 and 6),
are consistent with the model in which greater numbers of less
activated and less lytic CD8+ T cells are
developed at normoxic conditions, while hypoxia favors sustained
expansion of more lytic CD8+ CTL. Subsequent
experiments (Fig. 6) suggested that the higher IL-2 concentration found
in cell supernatants at 20% oxygen is not because there is more IL-2
production per cell at 20%, but because there is a higher number of
IL-2-producing cells at 20% than at 2.5% oxygen. However, the reasons
for higher IFN- accumulation are most likely due to both higher
number of T cells and a higher proportion of IFN--producing T cells
at 20% oxygen than at 2.5% oxygen.
Demonstration of much higher proportions of
TCRhighLFAhigh cells among
CD8+ 2.5 vs 20% oxygen (Fig. 7) provides a
possible explanation as to why these CTL are more lytic. The higher
surface density of TCR and LFA-1 recognition and cell adhesion
molecules at 2.5% oxygen may result in more lytic CTL, because these
surface molecules are important in lethal hit delivery by CTL due to
their role in CTL-target cell conjugate formation and lethal hit
delivery (35). It is much less clear why there are more
TCRhighLFAhigh
CD8+ T cells at 2.5% than at 20% oxygen. One
possibility is that a higher proportion of
TCRhighLFAhigh reflects
their better (preferential) expansion or survival at 2.5% oxygen than
at 20% oxygen. This, in turn, could reflect differences in cytokine
repertoire and concentrations in MLC conducted at different oxygen
tensions. For example, observations of increased VEGF accumulation at
2.5% oxygen (Fig. 5) suggest that the hypoxia-enhanced accumulation of
VEGF and/or of other yet to be determined gene products may create more
favorable conditions for the accumulation of
TCRhighLFAhigh CTL. Indeed,
it was reported that VEGF is able to induce increased levels of
LFA-1 molecules (36), which are important for CTL lytic
hit delivery. Interestingly, "hypoxic" CD8+ T
cells could not be enriched for studies using negative selection and
the AutoMACS separator by removing CD4 and B cells most likely due to
their more adhesive properties (data not shown). Future studies may
address in detail whether culturing cells under hypoxic
conditions change the ability of cells to participate in cell-cell
interactions and adhesion. In support of such an
interpretation are the observations of increased expressions of cell
surface adhesion molecules LFA-1 on the CD8 from 2.5% oxygen
(Fig. 7).
Thus, effects of hypoxia on T cells could be inhibiting (e.g., decrease
in IFN-, IL-2 secretion; see Fig. 5, C and D)
or enhancing (e.g., improvement of CD8+ CTL
development at 2.5 vs 20% oxygen, as shown in Figs. 3 and 4;
and of VEGF secretion, as shown in Fig. 5B). Other
functions, such as lethal hit delivery by CTL, are oxygen independent
(Figs. 2 and 3). The explanation of these opposite and cell
differentiation-dependent effects of oxygen tension could involve
effects of hypoxia-associated accumulation of extracellular adenosine
(21, 22, 37, 38, 39, 40) and activities of HIF-1 and HIF-2
(11). Indeed, differences between 1, 2.5, and 20% oxygen
tensions described here could be at least partially explained by the
patterns of HIF-1 expression (41, 42, 43, 44) and by
HIF-1-mediated changes in cellular response. It was shown that both
HIF-1 and HIF-2 activate transcription of hypoxia-inducible genes. The
common feature of these genes is the presence of HRE, and among
HRE-containing genes are VEGF, erythropoietin, and enzymes of
glycolytic pathway (11), which enable the switch of
ATP generation from oxidative phosphorylation in resting lymphocytes to
glycolysis in activated lymphocytes (45). It remains to be
determined in future studies whether HIF-1 or HIF-2, or both,
contribute to transcription of hypoxia-dependent genes in immune
cells.
These observations point to the need for better understanding of the
mechanisms of lymphocyte adaptation to hypoxia and of the role of HIF-1
and HIF-2 in these processes. Indeed, observations of the augmenting
effects of hypoxic exposure and of TCR triggering (VEGF vs IL-2 and
IFN-; see Fig. 5) do support the view that the overall effect of
hypoxia on T cell functions is to alter the proportion of HRE- and
non-HRE-containing cytokines. In contrast, genes that do not have
an HRE are expressed later and at levels which are higher in normoxic
conditions (20%) than at hypoxic conditions (2.5%). The accelerated
accumulation of VEGF in hypoxic conditions (Fig. 5) is best explained
by our recent findings that HIF-1 is an immediate-early response gene
(D. Lukashev, C. C. Caldwell, and M. V. Sitkovsky,
unpublished observations). Thus, the early presence of HIF-1 gene
product in activated T cells ensures earlier commencement of
transcription of hypoxia-dependent genes.
The observations of increased densities of TCR and LFA-1 on
CD8+ CTL at hypoxic conditions (Fig. 7) point to
the need in further studies of molecular mechanisms of this phenomenon.
Especially interesting are questions about the possible role of VEGF in
CD8+ CTL development. Even though VEGF has been
demonstrated to be a cause of increase of LFA-1 on NK cells
(36), it is not clear whether it was the direct effect of
VEGF or of other VEGF-induced cytokines on CD8+ T
cells in the experiment described in Fig. 5. Indeed, it is well
established that VEGF receptors are expressed mostly on endothelial
cells, and future detailed studies of VEGF receptor expression among
different immune cells may address the question as to what is the
mechanism of increase in the TCR and LFA-1 levels of expression on
CD8+ T cells in hypoxic conditions of MLC. Thus,
it is possible, although it remains to be further investigated, that
levels of TCR and LFA-1 in CTL are at least partially controlled by
VEGF levels and that the better lytic ability of CTL developed at
hypoxic conditions may be due to the functioning of hypoxia-dependent,
VEGF-mediated T cell recognition and cell adhesion molecules.
The data described demonstrate that the established routine conditions of lymphocyte culture (20% oxygen in the presence of reducing agent) are remarkably well suited to studies of immunologic specificities. Indeed, the importance of mimicking the in vivo redox potential in in vitro assays was well recognized in the original studies of lymphocytes in vitro. The use of a 7% oxygen mixture (46) as well as the addition of reducing agent 2-ME (29, 47) to incubations at 20% oxygen resulted in dramatic success in studies of Ag specificity in cellular immunologic assays in vitro. The observations of differences in lymphokine production between low oxygen and normoxic conditions could be helpful in studies of lymphokine-driven differentiation of Th1 vs Th2 and of Tc1 vs Tc2 cells (31), as well as in studies of T cell activation-dependent gene expression using DNA chip technology (48). The strong impact of in vitro oxygen tension on the outcome of T cell development adds yet another facet in considering the dramatically better than expected expansion of Ag-specific CTL in vivo using MHC class I/peptide tetramer detection assays (49, 50).
The question about the oxygen tension dependence of IFN- secretion
by human monocytes has also been addressed by Krieger et al.
(51), and together with our observations the data support
the model where immune cell development and functions proceed
differently at low hypoxia vs normoxia. Use of 1 and 2.5% oxygen
tensions, which were shown to be promoting the expression and functions
of HIF-1 (42), may represent a better approximation of the
hypoxic conditions in vivo. Furthermore, the observations reported here
suggest the use of hypoxic oxygen tensions in some in vitro assays to
better approximate in vivo conditions in studies of T cell functions.
The pH at 7.2 appears to be the most optimal and physiological way,
because direct measurements of pH in spleen of cats (23)
and rats (24) show the pH values of 7.20 and 7.21
respectively. The direct measurements of CO2 in
tissues were performed in studies of brain. It was shown that brain
pCO2 is normally 5%, yet even when the
brain becomes hypoxic, the pCO2 remains unchanged
(25, 26). These data suggest, in the absence of direct
measurements in spleen, that pCO2 is maintained
at relatively constant levels in tissues in vivo.
Our data also point to the need in further evaluation of cell culture
of hematopoietic stem cells and committed progenitors
(52). Indeed, the demonstration of differential effects of
hypoxia on 
vs T cells (data not shown) suggests that
similar lineage-specific effects could be taking place during the
expansion of marrow-repopulating progenitors in bone marrow.
Taken together, these observations may help in designing experiments to test the model of in vivo T cell expansion where more CD8+ CTL are formed in vivo in more vascularized tissue compartments with a sufficient network of capillaries and oxygen supply, while fewer CD8+ CTL but more potent CD8+ killers would accumulate in hypoxic areas.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: HIF, hypoxia inducible factor; HRE, hypoxia response element; KLH, keyhole limpet hemocyanin; VEGF, vascular endothelial growth factor.
Received for publication January 29, 2001. Accepted for publication September 21, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Genes Dev. 12:149.2-microglobulin-negative, MHC class I-deficient mice in response to immunization with tumor cells. J. Immunol. 152:2087.[Abstract]
(HIF-1) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions: its stabilization by hypoxia depends on redox-induced changes. J. Biol. Chem. 272:22642.: posttranscriptional regulation and conformational change by recruitment of the Arnt transcription factor. Proc. Natl. Acad. Sci. USA 94:5667.This article has been cited by other articles:
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  B. Sahaf, K. Atkuri, K. Heydari, M. Malipatlolla, J. Rappaport, E. Regulier, L. A. Herzenberg, and L. A. Herzenberg Culturing of human peripheral blood cells reveals unsuspected lymphocyte responses relevant to HIV disease PNAS, April 1, 2008; 105(13): 5111 - 5116. [Abstract] [Full Text] [PDF]
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 E. Piovan, V. Tosello, S. Indraccolo, M. Masiero, L. Persano, G. Esposito, R. Zamarchi, M. Ponzoni, L. Chieco-Bianchi, R. Dalla-Favera, et al. Differential Regulation of Hypoxia-Induced CXCR4 Triggering during B-Cell Development and Lymphomagenesis Cancer Res., September 15, 2007; 67(18): 8605 - 8614. [Abstract] [Full Text] [PDF]
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 C. C. Caldwell, J. Tschoep, and A. B. Lentsch Lymphocyte function during hepatic ischemia/reperfusion injury J. Leukoc. Biol., September 1, 2007; 82(3): 457 - 464. [Abstract] [Full Text] [PDF]
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