Download Lymphocyte protein synthesis is increased with

Clinical Science (2001) 101, 583–589 (Printed in Great Britain)
Lymphocyte protein synthesis is increased
with the progression of HIV-associated
disease to AIDS
Giuseppe CASO*, Peter J. GARLICK*, Marie C. GELATO†
and Margaret A. MCNURLAN*
*Department of Surgery, State University of New York at Stony Brook, Stony Brook, NY 11794-8191, U.S.A., and
†Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8191, U.S.A.
A
B
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T
R
A
C
T
HIV infection has been shown to affect lymphocyte function and to reduce lymphocyte
responsiveness in vitro to mitogenic stimulation, but little is known about lymphocyte
metabolism in vivo and how it is affected during the course of the disease. This study investigated
the metabolic activity of lymphocytes in vivo through the progression of HIV-associated disease.
Lymphocyte protein synthesis was measured with L-[2H5]phenylalanine (45 mg/kg body weight)
in healthy volunteers (n l 7), in patients who were HIV-positive (n l 7) but asymptomatic, and
in patients with AIDS (n l 8). The rates of lymphocyte protein synthesis [expressed as a
percentage of lymphocyte protein, i.e. fractional synthesis rate (FSR)] were not altered in HIVpositive patients compared with healthy controls (7.9p1.28 % and 9.1p0.53 %/day respectively),
but were significantly elevated in AIDS patients (14.0p1.16 %/day ; P 0.05). The serum
concentration of the cytokine tumour necrosis factor-α (TNF-α) increased with the progression
of the disease, and TNF-α levels were significantly higher in AIDS patients (6.81p0.88 ng/l)
than in healthy controls (3.09p0.27 ng/l ; P 0.05). Lymphocyte protein FSR was positively
correlated with serum TNF-α concentration (r l 0.55, P l 0.009) and negatively correlated with
CD4+ lymphocyte count (r lk0.70, P l 0.004). The elevation of lymphocyte protein synthesis
in AIDS patients suggests a higher rate of turnover of lymphocytes. This may be associated with
a generalized activation of the immune system, which is also reflected by the elevated serum
TNF-α concentration in the late stages of HIV-associated disease.
INTRODUCTION
HIV infection is associated with the progressive impairment of immune function. Several different abnormalities of lymphocyte function have been described,
affecting not only CD4+ T lymphocytes, which are the
main target of the HIV virus, but also other lymphocyte subpopulations. These include decreased lymphocyte
proliferation after mitogenic stimulation, reduced production of interleukin-2 and decreased expression of the
interleukin-2 receptor [1–7]. However, many of the
functional tests are carried out in vitro after isolating
lymphocytes from whole blood, and the results might
not be representative of lymphocyte metabolism in vivo.
Little is known about lymphocyte metabolism in
vivo and how it is affected in the different stages of HIVassociated disease.
The aim of the present study was to investigate
lymphocyte metabolism in vivo during the course of
HIV-associated disease, by assessing the rate of synthesis
Key words : AIDS, HIV infection, lymphocyte protein synthesis, L-[#H ]phenylalanine, stable isotopes.
&
Abbreviations : FSR, fractional synthesis rate ; HAART, highly active anti-retroviral therapies ; LBM, lean body mass ; TNF-α,
tumour necrosis factor-α.
Correspondence: Dr Giuseppe Caso (e-mail caso!surg.som.sunysb.edu).
# 2001 The Biochemical Society and the Medical Research Society
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G. Caso and others
of lymphocyte protein. Lymphocyte protein synthesis is
an index of cell metabolic activity, and has been shown to
be stimulated by treatments that induce activation and
proliferative responses of the immune cells, both in vitro
[8,9] and in vivo [10]. Lymphocyte protein synthesis
therefore represents an important metabolic marker of
immune cell turnover and activation, and it can be safely
measured in vivo with stable isotope techniques [10–12].
The effect of progression of HIV-associated disease
was assessed by comparison of rates of lymphocyte
protein synthesis in healthy subjects, patients with HIV
infection alone and patients with AIDS. The relationship
of the pro-inflammatory cytokine tumour necrosis
factor-α (TNF-α) to alterations in lymphocyte protein
synthesis was also assessed.
A preliminary report of this study has been published
previously in abstract form [13].
METHODS
Subjects
Seven healthy control subjects and 15 HIV-infected
patients participated in the study. Controls were recruited by local advertisement. HIV-infected subjects were
recruited from patients attending the AIDS Clinic at the
University Medical Center, Stony Brook. HIV-infected
subjects showing any signs of acute illness, opportunistic
infection or metabolic abnormalities, or those with
diagnosed malignancies or who had recently undergone
surgery, were excluded.
HIV-infected patients were divided into two groups,
HIV and AIDS. The HIV group (n l 7) included patients
who were HIV-positive but asymptomatic, with a CD4+
count of 400\mm$ and no history of opportunistic
infections. The AIDS group (n l 8) included patients
with a CD4+ count of 200\mm$ or who had experienced an AIDS-defining illness [14]. Once patients
were classified as having AIDS, they retained the classification even if CD4+ counts subsequently rose above
200\mm$. Consequently, at the time of the study two
patients in the AIDS group had CD4+ counts between
200 and 400\mm$. In the AIDS group three patients
experienced weight loss of 10 % compared with preillness body weight, and five had been treated previously
for opportunistic infections.
The study was carried out in the period 1994–1996,
before highly active anti-retroviral therapies (HAART)
had become the standard care of treatment of HIV
infection. Therefore none of the patients had been treated
with HAART before participating in the study. However, the majority of the HIV-infected patients in both
groups were taking medications, which were not discontinued before the study. These included antivirals
(two HIV and eight AIDS patients), prophylactic antibiotics or antimycotics (one HIV and six AIDS patients),
pain relievers (three HIV and two AIDS patients), and
# 2001 The Biochemical Society and the Medical Research Society
vitamin and mineral supplements (three HIV and four
AIDS patients).
The study protocol was approved by the Committee
on Research Involving Human Subjects at the State
University of New York at Stony Brook. Informed
written consent was obtained from all subjects involved
in the study.
Protocol
The study was carried out at the Clinical Research Center
(GCRC), University Hospital at Stony Brook. The
subjects were admitted at 17.00 hours and assessed
clinically. Lean body mass (LBM) was measured by dualenergy X-ray absorptiometry (DEXA, model DPX ;
Lunar Radiation, Madison, WI, U.S.A.). At 07.00 hours
the next morning, with the subject in the post-absorptive
state, two intravenous cannulas were inserted into contralateral forearm veins, and a blood sample was taken for
measurement of TNF-α concentration and HIV load.
Lymphocyte protein synthesis was then measured by
injecting a 2 % (w\v) solution of phenylalanine
(45 mg\kg body weight) (Ajinomoto, Tokyo, Japan)
containing 10 % L-[#H ]phenylalanine (MassTrace,
&
Woburn, MA, U.S.A.) over 10 min. Blood samples of
2 ml were drawn at various intervals over 90 min in order
to measure changes in plasma L-[#H ]phenylalanine
&
enrichment. A larger blood sample (30 ml) was then
taken at 90 min to determine isotope incorporation into
lymphocyte protein.
Isolation of lymphocytes
Blood for lymphocyte isolation was transferred into
heparinized tubes containing cycloheximide (final concentration 0.5 mM) (Sigma, St. Louis, MO, U.S.A.) in
order to prevent any further incorporation of the isotope
into cell protein. Lymphocytes were then isolated with a
modification of the method of Bøyum [15] which allowed
purification of lymphocytes from phagocytic cells and
platelets [16]. After sedimentation of red cells with 6 %
(w\v) dextran (Sigma) at room temperature for 30 min,
the leucocyte-enriched plasma was depleted of platelets
by addition of ADP (Sigma) [17]. Phagocytic cells were
then removed by preincubating the leucocyte-enriched
plasma with iron particles for 30 min at 37 mC.
Lymphocytes were then isolated by density gradient
centrifugation using Ficoll-Paque (density 1.077 g\ml ;
Pharmacia, Uppsala, Sweden) [15]. The lymphocyte
layers were removed and washed several times with 0.9 %
NaCl solution and then stored at k70 mC until analysis.
Measurement of lymphocyte protein
synthesis
Measurement of lymphocyte protein synthesis with
L-[#H ]phenylalanine was similar to that described pre&
viously [11,12,16]. Briefly, plasma phenylalanine enrich-
Lymphocyte protein synthesis in HIV infection
ment was determined after cation-exchange chromatography by monitoring the ions at m\z 336 and 341 of
the tertiary butyldimethylsilyl derivative on a VG MD
800 quadrupole gas chromatograph mass spectrometer
(Fisons Instruments, Inc., Beverly, MA, U.S.A.).
Lymphocyte protein was precipitated with cold perchloric acid (20 g\l) and washed extensively before
hydrolysis with 6 M HCl at 110 mC for 24 h. L[#H ]Phenylalanine enrichment in the hydrolysate was
&
measured after enzymic conversion of phenylalanine into
β-phenylethylamine, solvent extraction and derivatization [18]. The ions m\z 106 (mj2) and m\z 109
(mj5) of the heptafluorobutyryl derivative were
monitored under selective ion-recording conditions on
the same gas chromatograph mass spectrometer operated
under electron-impact conditions in splitless mode [19].
The lymphocyte protein fractional synthesis rate (FSR)
was calculated from the L-[#H ]phenylalanine enrich&
ment in the lymphocyte protein and the area under the
curve of the plasma free L-[#H ]phenylalanine (precursor)
&
enrichment, by using the formula [20] :
FSR (%\day) l 100iPt\A
where Pt represents the enrichment of phenylalanine in
protein at the end of the incorporation period and A is the
area under the curve of precursor enrichment against
time expressed in days. To the extent that protein
synthesized by lymphocytes might have been secreted
during the 90-min incorporation period, the rate of
lymphocyte protein synthesis will have been underrepresented.
Measurement of TNF-α and viral load
The serum concentration of TNF-α was determined on
duplicate frozen samples by using an ELISA kit with a
limit of detection of 0.5 ng\l (Quantikine High
Sensitivity ; R&D Systems, Minneapolis, MN, U.S.A.).
Viral load was measured by branched DNA hybridization (Quantiplex HIV-RNA assay ; Chiron, Emeryville,
CA, U.S.A.), as described previously [19].
Statistical analysis
The data are expressed as meanspS.E.M. Statistical
differences between means were analysed by two-tailed
Student’s t-tests for unpaired data. The correlation
between two sets of data was determined by linear
regression analysis. A P value of 0.05 was considered
statistically significant.
RESULTS
The characteristics and anthropometric measurements of
the subjects are summarized in Table 1. The three groups
were matched for age, which ranged between 25 and 48
Table 1
Characteristics of the subjects
Data are expressed as meanspS.E.M. Significant differences : *P
compared with control group.
0.05
Parameter
Control (n l 7)
HIV (n l 7)
AIDS (n l 8)
Male/female
Age (years)
Weight (kg)
Height (m)
Body mass index (kg/m2)
LBM (kg)
7/0
35p3
74p2
1.71p0.01
25p1
56.8p2.1
3/4
33p3
61p2*
1.66p0.01
22p1*
45.6p2.2*
7/1
37p2
68p5
1.75p0.04
22p1*
52.5p4.1
Table 2 Viral load, CD4+ lymphocyte count and TNF-α serum
concentration of the subjects in the control, HIV-positive and
AIDS groups
Data are expressed as meanspS.E.M. Significant differences : *P 0.01
compared with HIV group ; †P 0.01 compared with control group. ND, not
determined.
Parameter
Control
HIV
AIDS
CD4+ count (cells/mm3)
Viral load (equivalents/ml)
TNF-α (ng/l)
ND
ND
3.09p0.27
647p112
12 100p1430
4.94p1.23
134p45*
89 700p49 400
6.81p0.88†
years. The HIV-positive and AIDS groups had lower
body mass indices than controls (P 0.003). The average
weight and total LBM were also lower in the HIVpositive group than in controls (P 0.05), which might
reflect the greater number of female subjects included in
the HIV group (Table 1). However, the three groups
showed no differences in LBM when expressed as a
percentage of body weight (control, 76.5p2.2 % ; HIV,
75.1p2.0 % ; AIDS, 78.5p3.1 %).
Table 2 lists values for viral load, CD4+ lymphocyte
count and TNF-α serum concentration. In four out of six
patients in the HIV group and two out of eight in the
AIDS group, the viral load was below the value of
10 000 equivalents\ml of plasma, which represents the
lower limit of sensitivity of the assay used. For statistical
purposes, an arbitrary value of 10 000 was therefore
assigned to these patients. TNF-α serum concentrations
were significantly higher in the AIDS group (P l 0.007)
than in the controls.
Figure 1 shows the FSRs for lymphocyte protein in the
control, HIV and AIDS groups. The lymphocyte protein
FSR was 9.1p0.53 %\day (range 6.6–10.4 %\day) in the
control group, which was similar to that found in
the HIV-positive patients (7.9p1.28 %\day ; range 5.6–
15.3 %\day). The protein FSR in AIDS patients
(14.0p1.16 %\day ; range 8.8–19.1 %\day) was respectively 73 % and 54 % higher than that of HIV-positive
patients (P l 0.006) and controls (P l 0.02).
# 2001 The Biochemical Society and the Medical Research Society
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G. Caso and others
Figure 1 FSRs of lymphocyte protein in control subjects,
and in HIV-positive and AIDS patients
Horizontal bars represent means. Means labelled with the same lower-case letter
are significantly different (P 0.05).
When each value of lymphocyte protein FSR from the
three groups was plotted against the corresponding serum
TNF-α concentration, a significant positive correlation
was found (r l 0.55, P l 0.009) (Figure 2A). In HIVinfected individuals, both lymphocyte FSR and TNF-α
serum concentration were also negatively correlated with
the CD4+ count (Figures 2B and 2C). No correlation was
apparent between viral load and any of the other
parameters.
DISCUSSION
Progression of HIV infection to AIDS is characterized
by a depletion and dysfunction of CD4+ lymphocytes,
which ultimately results in severe impairment of immune
function. In the present study, lymphocyte metabolic
activity was assessed in vivo by measuring the rate of
lymphocyte protein synthesis using stable isotope methodology. The measurements were carried out in healthy
volunteers, in patients with asymptomatic HIV infection,
and in patients with AIDS. The results show that
lymphocyte protein synthesis rates in asymptomatic
HIV-infected individuals were comparable with those in
healthy controls, but that lymphocyte protein synthesis
was significantly increased in patients with AIDS,
suggesting increased activity of lymphocytes with the
progression of the disease.
The elevated rates of lymphocyte protein synthesis in
AIDS are unlikely to have been due to drug treatment.
Although there was some heterogeneity among patients
with respect to drug treatment, there was no obvious
relationship between lymphocyte protein synthesis rates
and particular classes of drugs.
Previous studies have described several immunological
abnormalities associated with HIV infection, such as
impairment of lymphocyte proliferation in vitro after
mitogenic stimulation and suppression of natural killer
activity [2,3,5–7]. In contrast with the findings of our
# 2001 The Biochemical Society and the Medical Research Society
Figure 2 Correlations between lymphocyte protein FSR and
serum TNF-α concentration (A) or CD4+ count (B), and
between serum TNF-α concentration and CD4+ count (C)
The correlations are all significant : (A) r l 0.55, P l 0.009 ; (B) r lk0.70,
P l 0.004 ; (C) r lk0.58, P l 0.025. #, Controls ; $, HIV-positive
subjects ; >, AIDS patients.
study, in which no change in lymphocyte activity was
detected in HIV-positive asymptomatic individuals,
defects in proliferative activity in vitro have been
observed in stimulated lymphocytes from patients in the
early stages of the disease [5,7]. These in vitro studies
were performed during maximal stimulation of isolated
lymphocytes with mitogens and, therefore, represent a
measure of the maximal potential responsiveness of
Lymphocyte protein synthesis in HIV infection
circulating lymphocytes to immune challenges. By comparison, the rate of protein synthesis of lymphocytes
assessed from in vivo labelling represents the prevailing
level of metabolic activity.
The in vivo data from the present study would suggest
that circulating lymphocytes in patients with asymptomatic HIV-associated disease are no more metabolically
active than those in healthy, uninfected individuals. The
rate of protein synthesis measured in the present study
includes protein that is turned over (synthesized and
degraded) within cells as well as protein synthesized for
the production of new cells. Increased lymphocyte
protein synthesis has been shown to be associated with
elevated lymphocyte proliferation [10].
With progression to AIDS, the rates of protein
synthesis in lymphocytes increased. This increase in
metabolic activity is consistent with the conclusions of
other studies examining the rate at which circulating
levels of lymphocytes increase following HAART
[21–23]. Because the CD4+ lymphocyte number increases
rapidly following the initiation of HAART, the HIV
pathogenesis of CD4+ depletion in AIDS has been viewed
as being the result of elevated cell destruction by the virus
and not of a failure of lymphocytes to regenerate [21–24].
Our study confirms the view of high lymphocyte
metabolic activity and proliferative activity in the late
stages of the disease.
Studies by McCune et al. [25] involving in vivo DNAlabelling of lymphocytes suggested that increased cell
turnover of lymphocytes contributed to the observed
increase in protein synthesis in patients with AIDS. The
study of McCune et al. [25] also reported increased
lymphocyte turnover in HIV-positive patients, which
appears to conflict with the present data on protein
synthesis. However, the criteria used to distinguish HIVpositive individuals from those suffering from AIDS
were different in the two studies. In the McCune et al.
study [25], five out of 11 HIV-positive subjects had
CD4+ lymphocyte counts of
200 cells\mm$ ; by this
criterion these subjects would, in the present study, have
been classified as having AIDS. In addition, based on
CD4+ lymphocyte number and viraemia, HIV-positive
patients in the study of McCune et al. [25] were at a later
stage of disease than the HIV-positive patients in the
present study.
There is a possibility that the higher rate of protein
synthesis observed in AIDS patients may reflect a
qualitative difference in the composition of the lymphocyte subpopulations in the peripheral blood with the
advancement of the disease. For example, it has been
observed that memory\effector-phenotype T-cell subpopulations have a higher turnover rate than naı$ vephenotype T cells [25]. An increase in the proportion of
memory\effector-phenotype T-cell subpopulations in
the peripheral blood with the progression of the HIV
infection may therefore contribute to the increased rate
of turnover of the total lymphocyte population [25],
which would also be reflected in a higher lymphocyte
protein synthesis rate.
In addition to increased lymphocyte protein synthesis,
increased rates of whole-body protein synthesis have also
been reported in AIDS patients [26]. Assuming that the
total mass of lymphocytic cells is approx. 1.5 kg, equivalent to 300 g of protein [27], it is possible to calculate
the increase in whole-body protein synthesis that is due
to the elevation in lymphocyte protein synthesis. The
reported increase in whole-body protein synthesis in
AIDS patients was approx. 27 µmol of leucine:h−":kg−"
[26]. Assuming body protein to be 8 % leucine [28], the
increase in lymphocyte protein synthesis could contribute approx. 20 % of the increase in protein synthesis
observed in the whole body.
The progression of HIV infection is commonly
associated with a general activation of the immune
system, which is reflected by elevated plasma levels of
several cytokines and immune-activation molecules
[29–33]. The higher rates of lymphocyte protein synthesis
in AIDS patients might therefore be the result of this
generalized activation of the immune system. As reported
by other authors [34–36], the serum concentration of
TNF-α was elevated in HIV-infected individuals. TNFα levels increased with the progression of the disease, and
were higher in AIDS patients than in healthy controls
(Table 2).
The conclusion that elevated lymphocyte protein
synthesis is the result of generalized activation of the
immune system is supported by the highly significant
inverse correlation between the rate of lymphocyte
protein synthesis and the serum concentration of TNF-α
(Figure 2A). It has been suggested that TNF-α might
have an important role in the pathogenesis and progression of HIV-associated disease [37,38]. TNF-α is one
of the cytokines that is produced by monocytes and
macrophages in response to the HIV virus [39,40], but it
has also been shown to enhance HIV replication in
infected cells [41–43] and to be positively correlated with
circulating HIV RNA levels in HIV-positive patients
[32]. Dysregulated production of TNF-α could therefore
re-inforce a vicious circle, by activating the immune
system on the one hand and enhancing HIV production
and CD4+ lymphocyte destruction on the other, thus
amplifying HIV infection and accelerating the clinical
progression of the disease. Consistent with this hypothesis are the results of the present study showing that
high TNF-α levels were associated with metabolic
activation of lymphocytes in patients with progressed
HIV-associated disease, and that both activation of
lymphocyte protein synthesis and elevated circulating
levels of TNF-α were associated with depletion of CD4+
cells (Figure 2).
In conclusion, the present study has investigated the
metabolic activity in vivo of lymphocytes in subjects at
# 2001 The Biochemical Society and the Medical Research Society
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G. Caso and others
different stages of HIV infection. Lymphocyte protein
synthesis rates in asymptomatic HIV-infected individuals
were found to be comparable with those in healthy
volunteers. In AIDS patients, however, rates of lymphocyte protein synthesis were increased by 54 %. The
enhancement of lymphocyte protein synthesis is consistent with a high rate of turnover of lymphocytes in
AIDS patients. This might be associated with a
generalized activation of the immune system, which is
reflected in elevated plasma levels of pro-inflammatory
cytokines, particularly TNF-α, in the late stages of HIVassociated disease.
11
12
13
14
15
16
ACKNOWLEDGMENTS
This study was supported in part by National Institutes
of Health grants DK 4931606 and M01RR10710. We
thank George A. Casella for his technical assistance.
17
18
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Received 12 March 2001/11 June 2001; accepted 13 June 2001
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