(Received for publication, July 5, 1995; and in revised form, September 22, 1995)
From the
Proliferative defects have been reported at the level of DNA
synthesis, even in T-lymphocytes from asymptomatic human
immunodeficiency virus type-1 (HIV-1
)
patients. Since purine and pyrimidine ribonucleotide availability is
crucial for proliferation, we compared the ability of HIV-1
and HIV-1
T-lymphocytes (>95% CD4
and CD8
) to activate de novo biosynthetic and salvage pathways following phytohemagglutinin
stimulation using
C-labeled precursors.
The striking
abnormality already detectable in asymptomatic patients' cells
was the impaired ability of CTP, UDP-Glc, and UTP pools to expand over
72 h (44-70% of control), although ATP and GTP pools and
responses were normal. In symptomatic patients, resting T-cells showed
markedly reduced pyrimidine pools (53-74% of control) with no
change following activation. Relatively normal ATP, GTP, and NAD pools
masked the same impaired response of de novo synthesis to
activation, with ATP and GTP being reduced by 50% at 48 h. Purine
salvage was more active than the control in unstimulated
HIV-1 cells.
This impaired de novo synthesis in HIV-1 T-lymphocytes severely
restricts the availability of ribonucleotides for vital growth-related
activities such as membrane expansion and strand break repair as well
as DNA and RNA synthesis. The data indicate that resting T-lymphocytes
from symptomatic patients survive through enhanced salvage, but that
stimulation induces metabolic cell death, and provide an explanation
for the activation-associated lymphocyte death seen in HIV-1
T-lymphocytes.
HIV-1 ()infection produces a series of abnormalities
that affect the hematopoietic system(1) . The most striking is
the profound depletion of CD4 cells, the main reservoir of the
virus(2) , but other alterations are seen, including general
lymphopenia, granulocytopenia, and anemia accompanied by lymphoid cell
depletion(1) . Furthermore, unexplained diarrheas and skin
problems are common complaints in infected patients(3) . The
hematopoietic cell precursors and basal membranes of the skin and
intestine are all cells with a high rate of division. Such uniform
abnormalities common to all dividing cells suggest a problem in normal
mitogenesis in vivo. This is supported by the diminished
capacity to produce bone marrow-derived colonies in vitro(4) and the failure of T-cells from infected patients to
proliferate after appropriate stimulatory
signals(5, 6, 7, 8) .
Failure of T-lymphocytes from infected patients to respond to recall antigens, mitogens, and T-cell receptor/TI stimulation can be detected in vitro by the inability of the stimulated cells to produce interleukin-2, to incorporate tritiated thymidine, and to complete their cell cycle(5, 6, 7, 8) . Defects in proliferation are seen in both CD4 and CD8 populations in infected patients(4, 5, 6, 7, 8) . Several explanations have been advanced for the failure of cells to proliferate after appropriate stimulatory signals. These include defects in antigen-presenting cells (6) or the lack of costimulatory molecules such as CD28, CD26 (a dipeptidyl peptidase that binds adenosine deaminase), or CD73 (a 5`-ectonucleotidase), all of which are decreased in HIV-1-infected patients(9, 10, 11) . Other possibilities are that structural and regulatory proteins of the virus, such as gp120, Tat, or Nef, might interfere with lymphocyte proliferation by binding to functional molecules(6, 12) . For example, gp120 binds to the CD4 molecule and can have the same effect as anti-CD4 antibodies. Tat protein binds to the CD26 molecule(13) , and Nef can bind to GTP and associate with a cellular serine kinase in T-lymphocytes(12, 13, 14) .
Two main
hypotheses have been put forward to account for T-cell death in HIV-1
infection. The first is programed cell death, or apoptosis, where even
unstimulated cells die within the first 24 h of culture. The nuclei of
the cells display electron-dense chromatin, and the DNA within the
nucleus is digested by an endogenous nuclease that cleaves chromatin
between individual nucleosomes while the membrane retains its
integrity(15) . Apoptosis can be reversed by the addition of
cytokines such as interleukin-2 and by fibroblast factors in the
cultures(16) . The second mechanism is the
activation-associated lymphocyte death, by which cells stimulated by
strong mitogenic stimuli such as phytohemagglutinin (PHA) or anti-CD3
die after 48-72 h in culture(7, 8) . In contrast
to cells dying from apoptosis, these cells leave the G phase of the cell cycle fully entering the blastic stage, defined
by an increase in size and the expression of activation markers such as
CD25 (interleukin-2 receptor) or Class II. These cells are nevertheless
unable to complete the cell cycle, their membrane disintegrates, and
they die, although no apoptotic bodies can be detected. This process
cannot be reversed in cultured cells from HIV-1
patients by the addition of interleukin-2, fibroblast factors, or
supernatants of noninfected cells activated by PHA(7) .
The
biochemical mechanisms associated with any of these processes have not
been defined, yet the development of effective antiviral therapy
demands an understanding of how infection changes the metabolism of
host lymphocytes in order to devise therapy selectively lethal to the
virus. Pharmacological agents currently on trial as antiviral and
immunosuppressive agents are purine and pyrimidine
analogues(17) . All require intact pathways of purine and
pyrimidine de novo synthesis or salvage for their
activation(18) . The normal human immune response also depends
on the activation of housekeeping genes, which results in the
stimulation of de novo purine and pyrimidine synthetic
pathways(43) , to provide the additional ribonucleotide
precursors necessary for growth-related activities including DNA
synthesis (the point at which activated HIV-1-infected T-cells from
asymptomatic patients reputedly perish). Altered activity has been
reported for purine and pyrimidine enzymes in lymphocytes from
HIV-1 subjects, but the focus has been only on
disrupted cells and enzymes catalyzing ribonucleotide degradation:
adenosine deaminase(19) , cytidine deaminase(20) , and
5`-ectonucleotidase(11) . Experiments comparing the integrated
activity of the synthetic and salvage routes in intact cultured
lymphocytes (which simulate the in vivo situation most
closely) are lacking.
This study stems from our earlier experiences
in the inherited immunodeficiencies: adenosine deaminase and
purine-nucleoside phosphorylase
deficiency(21, 22, 23) . The similarity
between the clinical consequences of adenosine deaminase deficiency and
AIDS, affecting the skin, intestine, and lung as well as the lymphoid
system, is remarkable. Indeed, the first adenosine deaminase-deficient
adults to be identified (two sisters in their thirties) were considered
to have a syndrome of ``non-HIV AIDS'' prior to referral to
us(23) . The experiments reported here were devised to
investigate whether a similar metabolic basis could be identified in
purine or pyrimidine metabolism to explain the mechanisms that prevent
T-lymphocytes of HIV-1 donors from finishing their
cycle. Sensitive high performance liquid chromatography (HPLC)
techniques, developed to differentiate between the purine
ribonucleotide and deoxyribonucleotide triphosphates that accumulate in
the above disorders(21, 23) , were used to measure
nucleotide pools(22) . In this report, we demonstrate that HIV
infection seriously impairs the capacity of T-lymphocytes to synthesize
the purine and pyrimidine ribonucleotide intermediates essential to
enable stimulated cells to complete their cycle(43) .
Figure 1:
A-C, time course showing the
number of viable lymphocytes present in an aliquot of T-lymphocytes
from an asymptomatic HIV-1 patient labeled with
anti-CD25 to detect the blastic population and analyzed with a Cytoron
absolute flow cytometer after culture in the presence or absence of
PHA. Shown is the absolute number of viable lymphocytes and
blasts/µl at day 1 prior to stimulation (A) and at day 4
in unstimulated (B) compared with stimulated (C)
lymphocytes. D-F, comparison of the number of viable
cells in the unstimulated (
) and PHA-activated (
) cultures
from HIV-1
controls (D) and asymptomatic (E) and symptomatic (F) HIV-1
patients. The results shown are the percentage of the initial
input at day 1. The mean plus range are
plotted.
Figure 2: Section of a high performance liquid chromatogram showing the separation of a standard mixture containing nine ribonucleotide and deoxyribonucleotide triphosphates (dNTPs). The chromatogram was recorded from 20 to 30 min at 254 nm (lower panel) with in-line photodiode array detection to show the UV spectra monitored simultaneously from 230 to 310 nm (upper panel). Peak maxima are also indicated. Note the clear separation of CTP, dCTP, ATP, dATP, UTP, dUTP, GTP, and dGTP. In this system, dTTP coelutes with GTP, as evident from the UV spectrum recorded from 230 to 310 nm, where the prominent shoulder characteristic of guanine-based compounds (see dGTP) is missing. The system described under ``Materials and Methods'' was developed to separate the 17 purine, pyrimidine, or pyridine-based compounds likely to be encountered in T-lymphocytes(43) . Any possible coelution of dTTP with GTP was not considered a problem since changes in the other dNTPs would be evident when the method could be optimized to separate and quantify all dNTPs.
Figure 3:
Mean ribonucleotide concentrations
(picomoles/10 cells) in T-lymphocytes from controls (left bars) and asymptomatic (center bars) and
symptomatic (right bars) HIV-1-seropositive patients. Shown
are uridine and cytidine nucleotides (A and B),
adenine and guanine nucleotides (C and D), and
pyridine nucleotides (E) measured on day 1 (D1) and
at 24 h (D2), 48 h (D3), and 72 h (D4) after
stimulation. UDPG, UDP-Glc.
Figure 4:
Incorporation of radiolabeled precursor
([C]glycine, 40 µM) of the de
novo pathway. Stimulated cells from controls and asymptomatic and
symptomatic HIV-1-seropositive patients were pulse-labeled for 2 h at
different intervals after stimulation: 0 h (D1), 24 h (D2), 48 (D3), and 72 h (D4). Shown is
[
C]glycine incorporation into adenine
nucleotides (A) and guanine nucleotides (B). Protein
content over 72 h is also shown (C).
The
noteworthy finding in symptomatic patients is that the relative
normality of purine pools in resting cells masks a grossly impaired
ability to respond to PHA, with adenine and guanine nucleotides
actually decreasing to 50% of control at 48 h (Fig. 3, C and D, right bars; p < 0.001).
The latter is consistent with the viability studies, which showed a
similar decline. NAD pools in unstimulated lymphocytes from symptomatic
patients also show no significant difference compared with controls,
but as for pyrimidines, the response to PHA is virtually absent (Fig. 3E; p < 0.001).
By
contrast, [C]glycine incorporation into ATP in
T-cells from symptomatic subjects was much lower. No radiolabel was
incorporated into GTP by symptomatic patients' cells, with
radiolabel being found only in IMP on day 4, indicating a block in the
conversion of IMP to GMP (Fig. 4, A and B).
These findings are in accord with the lack of any increase in protein
content (Fig. 4C) and the 50% fall in ATP and GTP
concentrations noted above (Fig. 3, C and D).
The results indicate that the inability of symptomatic patients'
cells to switch on genes activating both purine and pyrimidine de
novo synthetic pathways in response to PHA induces metabolic cell
death.
Figure 5:
Incorporation of radiolabeled precursors
(40 µM) of the purine salvage
([C]hypoxanthine) and pyrimidine salvage
([
C]uridine) pathways. Stimulated cells from
controls and asymptomatic and symptomatic HIV-1-seropositive patients
were pulse-labeled for 2 h at different intervals after stimulation: 0
h (D1), 24 h (D2), 48 h (D3), and 72 h (D4). Shown is [
C]hypoxanthine
incorporation into adenine nucleotides (A) and guanine
nucleotides (B) and [
C]uridine
incorporation into uridine nucleotides (C) and cytidine
nucleotides (D). UDPG,
UDP-Glc.
Pyrimidine salvage is clearly
much less active than for purines in unstimulated cells from all
groups, but incorporation of [C]uridine is
detectable and likewise most active in cells from symptomatic patients
at zero time. The pattern of uridine incorporation into uridine
ribonucleotides (Fig. 5C) following PHA stimulation in
asymptomatic patients' cells is normal, but is extremely poor in
symptomatic patients' cells, with virtually no conversion to
cytidine ribonucleotides (Fig. 5D).
These results
indicate that the ability of purine salvage to meet the requirements of
resting lymphocytes from symptomatic and asymptomatic HIV-1 individuals may explain the absence of marked differences in
purine ribonucleotide pools from controls. However, the reduced
incorporation of hypoxanthine into GTP relative to ATP by symptomatic
patients' cells suggests a block in GTP synthesis beyond the
level of the de novo intermediate IMP. The enhanced salvage of
uridine by resting cells from symptomatic patients is also consistent
with pyrimidine starvation. Although utilization of salvaged uridine
for CTP synthesis by all cells is poor, the lack of any conversion by
symptomatic patients' cells indicates a block at the level of CTP
synthetase.
Changes occurring in ribonucleotide pools of stimulated
HIV-1 and HIV-1
T-lymphocytes over
72 h are expressed here on an initial cell basis to highlight the
exponential expansion following PHA stimulation in control lymphocytes
and the striking aberrations induced by HIV-1
infection, namely (a) the early impairment in
asymptomatic patients' cells of the normal ability of pyrimidine
pools to expand in response to PHA; which is in sharp contrast to (b) the total inability of all pools in cells from symptomatic
subjects to respond to PHA, with ATP and GTP pools actually showing a
50% decline over 72 h; and (c) the enhanced salvage activity
in symptomatic patients' cells evident from the
C
incorporation studies, but reduced incorporation of hypoxanthine into
GTP and of uridine into CTP coupled with impaired de novo purine synthesis evident following PHA stimulation.
The
important question is how do the observed metabolic derangements relate
to the immunological abnormalities reviewed earlier? Although further
studies are clearly needed to provide a definitive answer, the
impairment of pyrimidine (and subsequently, purine and pyridine) pool
expansion in HIV-1 lymphocytes and the relationship of
this sequence of events to the severity of the clinical expression have
implications for several facets of the immune response other than the
mere synthesis of DNA and RNA.
Considering first the findings in
asymptomatic patients' cells, the selective impairment of
pyrimidine responses is in marked contrast to the disproportionately
greater expansion of pyrimidine pools in T-lymphocytes from healthy
donors. In the accompanying paper(43) , we propose that the
latter could be explained by the requirement to provide the additional
pyrimidine precursors essential for such growth-related activities as
protein glycosylation and membrane biosynthesis. Pyrimidine salvage may
be equally vital to such membrane-related processes. An additional role
for dCyd kinase in the salvage of dCyd not only for DNA, but also for
phosphatidylinositol synthesis via dCDP-choline and the corresponding
diacylglycerol intermediate was demonstrated recently(26) . The
high activity of dCyd kinase(26) , coupled with the low
activity of cytidine deaminase, in proliferating T-lymphocytes (27) would ensure the selective channeling of (d)Cyd to (d)CTP
synthesis. Consequently, the 37-fold induction of lymphocyte cytidine
deaminase reportedly induced by HIV-1 infection in a human H9 cell line (20) would severely restrict salvage of both Cyd and dCyd, with
subsequent impairment of CTP, DNA, and phosphatidylinositol synthesis.
Lymphocytes from HIV-1-infected subjects are known to
show aberrant inositol polyphosphate metabolism(28) . Such
HIV-1-induced CTP depletion could have equally important implications
for lipid synthesis in blasting lymphocytes. CTP depletion, accompanied
by arrest of CDP-choline biosynthesis, has been noted in cell lines
infected with other viruses(29) . Since CDP-choline and
phosphatidylinositols are active intermediates in membrane
biosynthesis, derangement of both these processes might explain the
HIV-1-associated membrane changes seen in asymptomatic patients'
cells(9, 10, 11) .
The poor response of
UTP pools and the reduced incorporation of radiolabeled glycine into
GTP in asymptomatic patients' cells are additional noteworthy
findings. Expansion of GTP as well as of UTP pools in stimulated
lymphocytes is equally important for membrane-related processes
associated with the additional growth-related demands for new
glycolipid and glycoprotein synthesis, such as formation of the
nucleotide dolichol phosphate-linked sugar intermediates essential for
the glycosylation of adhesion molecules (30) . The induction of
UTP and GTP depletion is known to be a key mechanism by which hexose
analogues exert their growth inhibitory or cytotoxic effects on human
cells(31) . The impaired expansion of UTP and UDP-Glc pools in
HIV-1 T-lymphocytes from asymptomatic subjects and the
poor response of GTP pools in symptomatic patients' cells would
similarly influence cell-surface topography and lectin binding, thereby
restricting mitogenic responses (28, 30, 32) . Altered glycosylation patterns
have been reported in CD45 (a molecule with tyrosine phosphatase
activity) in HIV-1
cells(33) .
The severe
impairment evident in the PHA responses of T-lymphocytes from
symptomatic patients in both de novo purine and pyrimidine
synthesis, coupled with the dramatic fall in ATP and GTP concentrations
and the inability to synthesize new protein, indicates that the cells
are metabolically dead. These findings are similar to those reported
for human blood lymphocytes incubated with deoxycoformycin and
deoxyadenosine to simulate adenosine deaminase
deficiency(34, 35) . Although viability, assessed by
trypan blue exclusion, was intact, the cells were otherwise
metabolically dead, being unable to synthesize proteins or RNA or to
respond to PHA stimulation(34) . The fact that ribonucleotide
pools in resting lymphocytes from our symptomatic subjects are not
elevated compared with controls is not consistent with the suggestion
that these cells are already stimulated and preactivated as reported by
others(2, 28) and thus cannot be restimulated.
Whether viral proteins or the virus itself is responsible is impossible
to tell. These cells are doomed. Interestingly, viral proteins might
also interfere with the mitogenic pathways of nonlymphoid cells. The
findings in these cells from symptomatic subjects mimic those following
preincubation of healthy T-lymphocytes with the glutamine antagonist
azaserine(43) , where PHA induced a decline in ATP and GTP to
50% of control and the response of all other pools was blunted or
nonexistent. Azaserine clearly inhibited several glutamine-dependent
reactions in stimulated T-lymphocytes from healthy controls that are
essential for pyrimidine, purine, and pyridine nucleotide
biosynthesis(36, 37) . The absence of responses to PHA
in symptomatic patients' cells here, coupled with the changes
already evident in lymphocytes from asymptomatic HIV-1 subjects discussed above (the blunted response in UTP, UDP-Glc,
and particularly CTP to stimulation plus the greater reduction in de novo GTP synthesis compared with ATP), is noteworthy. The
results suggest that HIV-1 infection, as well as restricting Cyd
salvage, may induce a similar defect affecting glutamine-dependent
reactions involving (a) the pyrimidine synthetic enzymes
carbamoyl-phosphate synthetase II and CTP synthetase, (b) the
purine biosynthetic enzymes formylglycine-amidine synthetase and GMP
synthetase, and (c) impairment of NAD synthetase.
Inhibition of NAD synthetase in HIV-1 cells could
provide an additional explanation for some of the abnormalities
observed, if as recent studies suggest, NAD depletion has implications
for the normal repair of DNA strand breaks in dividing rather than
resting lymphocytes(38) . NAD depletion (not evident in resting
HIV-1
lymphocytes from either asymptomatic or
symptomatic patients in this study, presumably due to enhanced purine
salvage) has been proposed to explain the lymphotoxicity to
unstimulated lymphocytes caused by the accumulation of dAdo in
inherited adenosine deaminase deficiency(35, 39) . NAD
is an essential substrate for chromatin-associated poly(ADP-ribose)
synthetase that is activated by DNA strand
breaks(38, 39, 40, 41) . The
triggering of programed cell death by the dAdo analogue
2-chlorodeoxyadenosine, recently developed as an immunosuppressive and
antineoplastic agent, has been related to the accumulation of DNA
strand breaks in both nondividing lymphocytes and monocytes, with
subsequent NAD depletion, inhibition of RNA synthesis, and eventual
cell lysis(41, 42) . In vitro studies
demonstrated that the initial DNA damage triggered the activation of a
Ca
/Mg
-dependent endonuclease,
resulting in an oligonucleosomal fragmentation pattern and
morphological changes typical of apoptosis(42) . The NAD
depletion evident in stimulated cells from the symptomatic
patients' cells here, coupled with the severe ATP depletion,
would provide an additional explanation for the mechanism by which
HIV-1 infection eventually induces lymphopenia and metabolic cell
death. Whether metabolic cell death and apoptosis are related in any
way requires further study.
The effect of HIV-1 infection on several metabolic aspects of lymphocyte proliferation demonstrated here could, as in adenosine deaminase deficiency, involve all cells undergoing active cell division and thereby explain the clinical findings in HIV-1-infected subjects relating to other cells of the hematopoietic system and involving cells of the skin and intestine as well. This study provides a metabolic explanation for the activation-associated cell death seen in T-lymphocytes from HIV-1-infected patients. The imbalance between the impaired de novo nucleotide synthesis and overstimulation of the salvage pathway explains the discrepancies in the number of viable cells from non-stimulated versus mitogenic stimulated cells after 72 h of culture. While unstimulated cells can replenish their nucleotide content through the salvage pathway, this is insufficient to enable stimulated cells to complete the cell cycle. Stimulated T-lymphocytes from asymptomatic patients and possibly some cells from symptomatic patients can initiate de novo synthesis to supply enough nucleotides to enable the cells to reach the blastic stage, but the nucleotide imbalance and membrane changes induced by the impaired pyrimidine nucleotide expansion make it impossible for these cells to complete the cell cycle. Such discrepancies could also explain why cells from HIV-1-infected patients are more susceptible to activation-associated cell death when stimulated by a strong mitogen, such as PHA, than when stimulated by milder mitogenic stimuli, such as pokeweed mitogen or protein A. Our data also explain how HIV-1 infection by inducing cytidine deaminase impairs pyrimidine salvage, which may be significant for nondividing cells as well(20, 27) .
Further studies are in progress to establish the level(s) at which HIV infection induces the observed irregularities and to pinpoint the enzyme(s) affected. Such knowledge has important implications for the design of new approaches to therapy.