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Infect Immun, June 1998, p. 2426-2433, Vol. 66, No. 6
Laboratory of Cellular Physiology and
Immunology, Rockefeller University, New York, New
York1;
Division of Mycobacterial
Research, National Institute for Medical Research, London, United
Kingdom2; and
Department of Medicine,
University of Cape Town, Cape Town, South Africa3
Received 7 July 1997/Returned for modification 3 September
1997/Accepted 28 February 1998
Administration of low-dose recombinant human interleukin 2 (rhuIL-2) in combination with multidrug chemotherapy to patients with
multidrug-resistant tuberculosis (MDR TB) induces measurable changes in
in vitro immune response parameters which are associated with changes
in the clinical and bacteriologic status of the patients. To determine
the molecular basis of these changes, we have used semiquantitative
reverse transcriptase-initiated PCR (RT-PCR) and differential display
technology. During rhuIL-2 treatment of MDR TB patients, decreased
levels of gamma interferon (IFN- Multidrug-resistant tuberculosis
(MDR TB) is a devastating disease from which only 55% of
immunocompetent patients recover after a prolonged course (a minimum of
24 months) of multidrug chemotherapy, while 22% ultimately die of TB
(10). Patients who are coinfected with human
immunodeficiency virus have a 50% response rate to antituberculosis
chemotherapy, with 50% mortality due to TB (38). A
potential means of improving patient response and survival is to
combine optimal chemotherapy with pharmacologic augmentation of host
immunity (immunomodulation). Recently, interleukin-2 (IL-2) has
received increasing attention as an immunomodulatory drug in human
infectious diseases. This cytokine is a pivotal regulator of
cell-mediated immunity and has been shown to induce the proliferation
and differentiation of lymphoid cells (36).
We have previously reported that patients with MDR TB treated with
low-dose recombinant human IL-2 (rhuIL-2), in combination with
optimized antituberculosis chemotherapy, show evidence of an enhanced
antimicrobial response (21). In addition to decreased sputum
bacillary load and decreased mycobacterial viability observed in
response to rhuIL-2 adjunctive therapy, some of the patients experienced amelioration of symptoms and an improved lung radiologic picture (21, 22). These improvements were associated with an
IL-2-induced increase in peripheral blood leukocyte CD25 and CD56
expression. In addition, we observed increased leukocyte proliferation
in vitro in response to either purified protein derivative of
tuberculin (PPD) or exogenously added IL-2, as well as an increase in
lymphokine-activated killer cell-mediated cytotoxicity (21).
However, the cellular and molecular events regulating these changes are
as yet unclear.
The availability of MDR TB patients who are responding clinically and
bacteriologically to treatment with an immune modulator such as rhuIL-2
provides a novel opportunity to investigate the molecular and cellular
events involved in the Th1-type protective immune response. It is
important to establish in vitro correlates of protective immunity so
that the prognosis of infected individuals and the effectiveness of
immune intervention strategies can be rapidly assessed; by
investigating IL-2-induced changes in the immune status of patients, it
might be possible to identify those changes which correlate with a
favorable outcome.
In this study we have used a combination of semiquantitative reverse
transcriptase PCR (RT-PCR) and differential-display technology to
investigate the molecular basis of observed clinical and in vitro
immunologic changes in MDR TB patients receiving adjunctive rhuIL-2
treatment to gain insight into how perturbations in immune activation
might affect bacterial clearance and clinical status. By investigating
changes in gene expression in skin biopsies taken from sites of
delayed-type hypersensitivity (DTH) reactions to mycobacterial antigens
and from peripheral blood mononuclear cells (PBMC), we have begun to
identify genes whose expression varies during rhuIL-2 therapy and which
may contribute to the specific immune response to mycobacteria and to
antimicrobial activity.
Patient population.
Fourteen human immunodeficiency
virus-seronegative hospitalized patients in Cape Town, South Africa,
with culture-confirmed MDR TB and currently receiving optimized
antituberculosis chemotherapy enrolled in this clinical study. One
group of patients (n = 10) were given twice-daily
intradermal injections of 12.5 µg (225,000 IU) of rhuIL-2 Aldesleukin
Proleukin (Chiron Corporation, Emeryville, Calif.) for 30 days as
described previously (21). A group of matched MDR TB
patients (n = 4) who were treated with a placebo and
not IL-2 were included as controls (Table
1). The study was approved by the
research institutional review board of the Rockefeller University
Hospital, New York, N.Y., and by the ethics committee of the University
of Cape Town, Cape Town, South Africa. All patients gave written
informed consent.
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Differential Gene Expression in Response to
Adjunctive Recombinant Human Interleukin-2 Immunotherapy in
Multidrug-Resistant Tuberculosis Patients
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) mRNA in peripheral blood
mononuclear cells (PBMC) relative to baseline levels were observed.
However, at the site of a delayed-type hypersensitivity (DTH) response
to purified protein derivative of tuberculin (PPD), the expression of
cellular IFN- and IL-2 mRNAs was increased during rhuIL-2 therapy.
Levels of other cytokine mRNAs were not significantly affected by
rhuIL-2 administration. Using differential-display RT-PCR, we
identified several genes expressed at the DTH skin test site which were
up- or down-regulated during rhuIL-2 treatment. Cytochrome oxidase type
I mRNA was increased in response to rhuIL-2 therapy relative to
baseline levels, as was heterogeneous nuclear ribonuclear protein G
mRNA. CD63, clathrin heavy chain, and 
-adaptin mRNAs, all of which
encode proteins associated with the endocytic vacuolar pathway of
cells, were also differentially regulated by rhuIL-2 administration.
The differential effects of IL-2 were confirmed in vitro by using PBMC
obtained from PPD-positive individuals stimulated with
Mycobacterium tuberculosis and IL-2. The differential
expression of genes may provide a surrogate marker for leukocyte
activation at a mycobacterial antigen-specific response site and for
the development of an enhanced antimicrobial response which may result
in improved outcomes in MDR TB patients.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Patient characteristics and treatment
Peripheral blood collection. Blood was collected from MDR TB patients at three time points during the study: before rhuIL-2 administration (prestudy), after completion of 14 days of rhuIL-2 administration (day 15; midstudy), or 7 days following the last rhuIL-2 injection (day 37; poststudy). For control patients, blood was collected at equivalent time points. Blood was also collected from four normal PPD skin test-positive donors. PBMC were isolated by using Ficoll-Hypaque gradients as described below (21).
PBMC stimulation in vitro. (i) PBMC preparation.
Human PBMC
were obtained from heparinized blood by centrifugation through a
Ficoll-Hypaque gradient. Freshly isolated PBMC (106/ml)
were stimulated with 50 µg of heat-killed sonicated
Mycobacterium tuberculosis H37Ra (Difco, Detroit, Mich.) per
ml and/or 100 IU of rhuIL-2 (Chiron Corporation) per ml in RPMI (GIBCO,
Grand Island, N.Y.) containing 10% pooled A-positive human serum
(R10). Cells were harvested at 0, 2, 6, 24, 48, 72, and 96 h
poststimulation. For proliferative assays, 105 stimulated
cells/well were incubated overnight with 1 µCi of [3H]thymidine before harvest on glass fiber filters and
scintillation counting. Culture supernatants were collected for
evaluation of gamma interferon (IFN-) levels by enzyme-linked
immunosorbent assay (Medgenix, Fleurus, Belgium) according to the
manufacturer's instructions.
(ii) Preparation of mycobacterial antigen.
M.
tuberculosis H37Ra (Difco) was used for stimulation of PBMC in
vitro. Desiccated heat-killed mycobacteria (100 µg/vial) were
suspended in R10 medium to 10 µg/ml, vortexed twice for 1 min each
time, and incubated at 4°C for 1 h. The bacterial suspension was
sonicated three times for 20 s each at an output of 2 at 40% duty
cycles with a probe sonicator (Heat Systems, Ultrasonics Inc.,
Farmingdale, N.Y.) and then centrifuged at 800 × g for 3 min, aliquoted into 1-ml tubes, and frozen at 
70°C until used.
DTH to mycobacterial antigens. Skin tests for DTH responses to PPD (5 tuberculin units/test; Connaught Laboratories Limited, Ontario, Canada) were injected intradermally on the forearm at the start of the study (prestudy) and on day 28 of rhuIL-2 adjunctive therapy (or at equivalent time points for control patients). The local skin test response was evaluated at 24-h intervals, and 4-mm punch biopsies of the skin test site were obtained at 48 h postinjection. Punch biopsies were immediately frozen in liquid nitrogen.
RNA extraction and semiquantitative RT-PCR for cytokine mRNAs. RNA was extracted from PBMC and from homogenized skin biopsies by using RNAzol B (Tel-Test, Inc., Friendswood, Tex.). Total RNAs from six of the patients treated with rhuIL-2 and four patients treated with the placebo were reverse transcribed and PCR amplified for cytokine mRNA and control mRNA by using procedures and primers described previously (20, 21). Following Southern hybridization with radiolabeled probes, PCR-amplified cDNA was quantitated by using radioanalytic imaging and normalized to the constitutively expressed glyceraldehyde 3-phosphate dehydrogenase cDNA for RNA quantity control. mRNA [poly(A+)] was isolated from in vitro-stimulated PBMC obtained from the blood of normal PPD-positive donors by using the Quick Prep micro mRNA purification kit (Pharmacia Biotech, Piscataway, N.J.) according to the manufacturer's instructions.
DDRT-PCR. Total RNAs from PBMC or homogenized skin biopsies of six of the patients treated with IL-2 and the four placebo-treated patients were used for differential-display RT-PCR (DDRT-PCR). RNA was treated with DNase (Promega, Madison, Wis.), after which 0.5 µg of RNA was reverse transcribed by using one of the anchored primers (DD1 [T12CA], DD2 [T12CG], or DD3 [T12CC]). Replicate reactions with enzyme omitted or with only H2O instead of RNA were set up for negative controls.
Following reverse transcription, PCRs with both anchored primers and arbitrary primers (OPA14 [TCTGTGCTGG], OPA18 [AGGTGACCGT], OPA20 [GTTGCGATCC] [3, 24], or AP4 [GCTCTTTGTC] [28]) and
-35S-dATP (NEN, Boston, Mass.) were set up. The PCR
conditions were 92°C for 50 s, 40°C for 1.5 min, and 72°C
for 1 min, for 35 cycles.
Amplification products were electrophoresed on a 6% denaturing
polyacrylamide gel, dried without fixation, and exposed for 1 to 4 days
to BioMax MR scientific imaging film (Eastman Kodak Co., Rochester,
N.Y.). Bands which appeared to be consistently differentially expressed
in multiple patient RNA isolations were excised from the dried gel,
eluted for 3 h at 65°C in Tris-EDTA, and reamplified by PCR with
the appropriate primer sets, low-stringency conditions, and 30 cycles
of 94°C for 30 s, 52°C for 40 s, and 72°C for 1 min.
PCR products were then electrophoresed on a single-strand conformation
polymorphism gel for analysis of product purity (26).
Cloning and sequence analysis. The reamplified cDNA was subcloned into a TA pCRII or a pCR2.1 vector (Invitrogen, San Diego, Calif.) and sequenced (Sequenase version 2.0 DNA sequencing kit [U.S. Biochemicals] or DNA Stretch Sequencer [Perkin-Elmer/Applied Biosystems], with SP6 and T7 or M13 and T7 promoter primers, respectively). The nucleotide sequences obtained were compared with known sequences by searching the GenBank, EMBL, and EST databases with the BLASTN search program of the National Center for Biotechnology Information Blast Network (26 March 1996) (1).
Northern blots. Total RNA at 20 µg/lane or poly(A)+ RNA at 1 µg/lane was subjected to electrophoresis by the standard formaldehyde protocol (34). RNA was transferred to a Zeta-Probe membrane (Bio-Rad, Hercules, Calif.) in 50 mM NaOH and UV cross-linked. Membranes were probed with 32P-labeled antisense RNA transcribed in vitro (Promega) after ligation of an optimized T7 promoter (Ambion, Austin, Tex.) to PCR-amplified cDNA.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Patients with MDR TB were enrolled in this study and assigned to rhuIL-2 treatment or control study groups while maintaining their antimycobacterial drug regimen (Table 1). To monitor the effects of IL-2 on leukocyte gene expression, PBMC were isolated from the blood of all patients at baseline upon study entry (prestudy), at day 15 of rhuIL-2 treatment (or the corresponding time point for controls) (midstudy), and 7 days following the last rhuIL-2 injection (or the corresponding time point for controls) (poststudy). In addition, PPD skin tests were performed for both groups of patients at the time of study entry and again after 28 days of rhuIL-2 injections (or at similar time points for control patients). A biopsy was taken from these tuberculin-reactive skin test sites at 48 h postinjection. Sets of two biopsies (baseline and 28 days) (of about the same size) were selected from six IL-2-treated patients and the four control patients not treated with IL-2. These biopsies and blood samples from the same 10 patients were used for cytokine mRNA analysis.
Cytokine gene expression in PBMC.
Southern blotting of
serially diluted RNAs from PBMC (baseline, 15 days, and 37 days) of
four control patients was performed. Total RNA from the samples was
amplified by RT-PCR and hybridized to radiolabeled probes specific for
IFN- and IL-2 mRNAs. In PBMC obtained from all control patients
tested, IFN- mRNA was readily measurable at all time points. IL-2
mRNA levels were lower and were detectable in PBMC only at the highest
level of input RNA (50 ng of total RNA per reaction) (not shown).
, IL-2, tumor necrosis factor alpha (TNF-), IL-4, IL-10, IL-12, and the T-cell marker CD3
. IFN- mRNA levels in PBMC decreased at the mid- and poststudy time
points relative to the pre-IL-2 treatment time point (Table 2). Levels of the T-cell surface marker
CD3 mRNA did not change significantly during rhuIL-2 adjunctive
therapy (Table 2). Since T cells are a major source of IFN-, the
ratio of IFN- mRNA to CD3 mRNA was calculated to evaluate the
changes in IFN- mRNA expression on a per-T-cell basis. A decrease in
the level of IFN- mRNA expression per T cell during and immediately
following rhuIL-2 therapy was observed (Table 2). This change was not
observed in the control patients not treated with rhuIL-2. Because the expression of IL-2 mRNA in patient PBMC was very low, we did not compare the effects of rhuIL-2 treatment on the expression of this
cytokine mRNA in the two study groups.
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mRNA expression was undetectable in PBMC of three of six
rhuIL-2-treated patients at all time points during the study and was
unchanged by rhuIL-2 therapy in PBMC of four of six patients in whom
low levels of TNF- mRNA were detected at baseline (data not shown).
Levels of IL-10 mRNA in patient PBMC at the pre-, mid-, and poststudy
time points were low or below the limit of detection in all patients at
all time points. IL-12 mRNA and IL-4 mRNA were not detected in PBMC of
patients.
Cytokine gene activation in response to PPD administration in the
skin.
The importance of measuring cytokine gene activation at the
site of mycobacterium-specific immune reactivity was underscored by the
observation that mRNA expression for the Th1 cytokines IFN- and IL-2
is generally higher in this specific skin test site than in cells of
the peripheral blood of the same patients. Also, IFN- mRNA
expression is higher than IL-2 mRNA expression in the cells of the DTH
response to PPD at 48 h (not shown). The expression of cytokine
genes, including those for IL-2, IFN-, IL-1, IL-4, IL-10, IL-12,
and TNF- at the PPD site, and the influence of rhuIL-2
administration on cytokine gene expression at this site were assessed
for six MDR TB patients treated with rhuIL-2. In parallel, levels of
IFN- and IL-2 mRNAs in PPD biopsies of four control patients were
quantitated.
were increased (1.3- and 2.5-fold, respectively, and 3.5- and 3.0-fold,
respectively, on a per-T-cell basis) during rhuIL-2 therapy relative to
the prestudy timepoint (Fig. 1). When calculated as the percent change from baseline levels of gene expression, IL-2 and IFN- mRNA levels increased to 175 and 200%, respectively, in response to rhuIL-2 treatment (Table 2). In control
patients the expression of IL-2 and IFN- mRNAs was not increased
(115 and 110%, respectively). IL-4 mRNA was not detected in the
biopsies of five of six patients and was measurable at a very low level
in RNA from only one patient biopsy (not shown).
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, IL-10, IL-12, and TNF- mRNAs (Table 2). An
increase in the level of mRNA for IL-10 was observed in cells of the
PPD site during rhuIL-2 therapy. IL-1 mRNA expression was not
changed in the biopsies of most patients tested. Similarly, IL-12 mRNA
and TNF- mRNA expression at the site of the response to PPD
administration did not change from the pretreatment to 28-day rhuIL-2
treatment time points. These results taken together indicate that
administration of rhuIL2 resulted in changes in IL-2 and IFN- mRNA
gene expression and that these changes differed at sites of specific
antigen stimulation compared to responses in the peripheral blood.
Differential-display analysis of mRNAs isolated from biopsies of PPD skin test sites. In order to gain understanding of changes in expression of other genes which might occur at sites of antigenic stimulation, we used DDRT-PCR, a technique which permits the recognition of changes in expression of unselected genes (24, 25). PCR amplifications of RNA from PPD skin tests done before and during rhuIL-2 administration for six patients (or at the corresponding time points for four control patients not treated with rhuIL-2) were performed with 12 primer combinations on each RNA sample. A comparison of cDNA displays generated from RNAs isolated from patient biopsies obtained before and during rhuIL-2 administration (28 days) and from control patient biopsies obtained at similar time points is shown in Fig. 2. As can be seen from Fig. 2, several cDNA bands are differentially regulated in response to IL-2.
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-adaptin mRNA, also within the coding region (GenBank accession no.
M34175) (32). cDNA B7a matched the sequence for human
heterogeneous nuclear ribonuclear protein G (hnRNP-G) mRNA with 97%
identity in the translated sequence over 137 bp (GenBank accession no. Z23064) (37). B7b was 99% identical over 135 bp within the coding region to human cytochrome oxidase subunit I mRNA (GenBank accession no. M10546) (35). B17 matched with 100% identity over 154 bp and cDNA B20 matched with 98% identity over 240 bp the
sequence of GenBank accession no. D21260, human mRNA for the KIAA00334
gene, which is similar to rat clathrin heavy-chain mRNA
(29). cDNA bands B23, B26, and B27 were found to be
homologous to sequences deposited in the expressed sequence tag
database created by the WashU-Merck EST Project (18). Band
B23 was 97% identical over 151 bp to human cDNA clone 44827 (GB_EST
H06717). Sequence B26 was 99% identical to human cDNA clone 246179 (GB_EST3: N55516). Band B27 was 98% identical over 146 bp to human
cDNA clone 83610 (GB_EST6: T61071).
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Differential-display analysis of mRNAs isolated from PBMC.
DDRT-PCR was performed on RNA isolated from PBMC obtained from the same
patients before IL-2 treatment, during IL-2 administration (15 days),
and after IL-2 treatment (37 days), similarly to the analysis performed
on the biopsy sites (see above). An example of the cDNA display gels
generated is shown in Fig. 3. While many PBMC genes were differentially expressed in response to rhuIL-2 administration, not all of the genes identified to be regulated at the
PPD site were also similarly regulated in the PBMC (Table 3). Of the
nine genes shown to be up- or down-regulated at the PPD site in
response to rhuIL-2 treatment, those for CD63, -adaptin, hnRNP-G,
and cytochrome oxidase type I were not found to be modified in PBMC
during or after rhuIL-2 administration. Clathrin heavy chain, which was
up-regulated during rhuIL-2 treatment in patient biopsies (B17), was
independently shown to be differentially expressed in PBMC (B20), where
it was down-regulated at the mid-IL-2 treatment time point and
undetected at the post-rhuIL-2 treatment time point. Three sequences
with homology to human cDNA genes, B23, B26, and B27, were similarly
down-regulated in the blood and at the PPD response site. Band B24,
which was 99% homologous over 140 bp to human cDNA clone 259934 (GB_EST N32924), was not identified at the PPD site but was found to be
differentially regulated in PBMC (Table 3).
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IL-2-induced differential expression of genes in PBMC stimulated in
vitro with M. tuberculosis.
To confirm the specific
response to M. tuberculosis and IL-2, PBMC isolated from
normal PPD-positive donors were stimulated in vitro with either
M. tuberculosis alone or M. tuberculosis in the
presence of rhuIL-2. RNA was isolated from the PBMC at 2, 3, and 4 days
after initiation of cultures, and DDRT-PCR was carried out.
Supernatants from the cultures were collected for IFN- level
determination. Three of the cDNA bands observed to be up-regulated at
the PPD site following IL-2 treatment were selected for analysis (B7a,
B7b, and B17). All three bands were up-regulated in vitro in response
to M. tuberculosis and were further up-regulated when IL-2
plus antigen was added to the cultures (Table
4). The degree of differential expression
varied among the bands. Differential expression was usually more
pronounced on day 4. In addition, IFN- production was induced by
exposure of the cells to M. tuberculosis and further
increased by the addition of IL-2 (Table 4). No IFN- was induced by
IL-2 treatment alone. The genes for hnRNP-G and clathrin heavy chain,
both expressed at relatively high levels in resting cells, were
evaluated from total cellular RNA. However, the gene for cytochrome
oxidase I was not expressed at high enough levels in total RNA and
required isolation of mRNA for quantitation of differential expression. The gene for cytochrome oxidase I (B7b) appeared to be most sensitive to differential regulation in the presence of IL-2 (Table 4 and Fig.
4). A similar pattern was reproducibly
observed in cells of the same donor, and in different donors, although
the degree of expression varied from donor to donor.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In our previously reported studies, patients with MDR TB received low-dose rhuIL-2 in combination with optimized multidrug antituberculous therapy. We observed immunologic responses to this adjunctive treatment which were associated with clinical, bacteriologic, and radiologic changes. Changes included decreased sputum bacterial load and viability, improved chest X-ray results, and decreased symptoms in approximately 60% of patients (21, 22). During rhuIL-2 administration, there was enhanced in vitro lymphocyte proliferation to mycobacterial antigens, augmented leukocyte proliferative activity to IL-2 in limiting-dilution assay in vitro, increased lymphokine-activated killer activity, and expanded numbers of CD25+ (low-affinity IL-2R) and CD56+ (NK) cells in the peripheral blood. In order to define the underlying cellular and molecular bases of these changes, we have assayed the changes in transcriptional activity of mononuclear cells of the blood and in the biopsies of PPD-reactive skin test sites at time points during and following rhuIL-2 adjunctive therapy for comparison with levels measured at the pre-IL-2 time point.
As observed in our previous study (21), we report here that
levels of IFN- mRNA in PBMC generally decreased during rhuIL-2 administration coordinately with improvement in patient symptoms. Other
cytokine mRNA levels in the cells of the peripheral blood of these
patients, including IL-1, IL-2, IL-4, TNF-, IL-10, and IL-12 mRNA
levels, remained unaffected by rhuIL-2 administration. A different
pattern was observed in the PPD skin test sites, where we observed
increased expression of IL-2 and IFN- mRNAs during rhuIL-2 therapy.
This effect was selective, since the expression of other cytokines,
including IL-1, IL-12, and TNF-, was not significantly affected.
In our pilot study (21) we had reported that cytokine mRNA
expression levels in cells at the PPD site were not significantly
influenced by 14 days of adjunctive rhuIL-2 therapy. In our current
study we performed the second PPD injection after 28 days of rhuIL-2
therapy and took a biopsy from the site on day 30 of treatment. Thus,
the effects of IL-2 were evaluated in the present study following a
longer period of rhuIL-2 treatment. It is possible that the additional
14 days of daily rhuIL-2 administration may have significantly modified
the host response to antigen stimulation in the skin.
IL-2 and IFN- have been demonstrated to play major roles in the
development of cell-mediated immunity (CMI) to mycobacterial infection.
In patients infected with Mycobacterium leprae, expression of IL-2 and IFN- predominates in the skin lesions of tuberculoid leprosy patients with an active CMI response, whereas these cytokines are not expressed in the skin of lepromatous leprosy patients, who do
not have protective CMI to the infecting bacilli (41). Moreover, IFN- production is enhanced at the site of disease in the
pleural fluid in patients with tuberculous pleuritis (2). Since this manifestation of tuberculosis is usually self-resolving concomitant with the emergence of CMI (15), these
observations provide evidence for the contribution of the Th1-type
cytokine IFN- in protective immunity to mycobacterial infections.
The reduced levels of IFN- mRNA seen in PBMC, in contrast to the
increase seen at the PPD site, suggest that in response to rhuIL-2
administration, mycobacterium-specific T cells home to sites of
antigenic stimulation, such as the infected lung or the PPD skin test
site, where they become primed or activated to produce elevated levels
of Th1 cytokines. These results reinforce the importance of
investigating immune responses at the sites of infection, particularly
when attempting to identify correlates of protective immunity, since
responses in the peripheral blood may not truly reflect
antigen-specific responses that are occurring elsewhere.
IFN- is a major contributor to macrophage activation (16,
17) and to the induction of two important antimycobacterial pathways, leading to the generation of reactive oxygen intermediates and L-arginine-derived nitric oxide (NO) (7, 8, 11,
30, 33, 40). Despite the evasive mechanisms employed by
mycobacteria to block the production and resist the toxic effects of
oxygen radicals as demonstrated in vitro (4, 8, 12, 13), the respiratory burst generated by IFN--activated phagocytes may play an
important role in the control of M. tuberculosis in vivo. This pathway might contribute to the rhuIL-2-induced bacteriologic effects seen in this study.
The use of DDRT-PCR to identify novel genes which may be IL-2 regulated
at the site of an antigen-specific response (the PPD skin test site)
has provided some intriguing findings. Three of the differentially
expressed genes, those for CD63, clathrin heavy chain, and -adaptin,
are associated with the endocytic vacuolar pathway of cells. CD63 is a
lysosomal membrane glycoprotein and has been shown to be present on the
membranes of macrophage phagosomes containing dead M. tuberculosis bacilli and not present on phagosomes containing live
organisms (9). Thus, the molecule appears to be associated
with a more mature vacuole. Clathrin heavy chain is a structural
component of coated pits and vesicles which are responsible for
selective endocytosis of ligand-bound plasma membrane proteins,
including receptors for various extracellular ligands. Clathrin-coated
vesicles are also associated with the trans-Golgi network and
facilitate the delivery of newly synthesized lysosomal enzymes to a
prelysosomal compartment (6). -Adaptin is a subunit of
the plasma membrane adapter complex which functions in binding to the
cytosolic domains of certain receptors, leading to the assembly of
clathrin (31). Differential regulation of genes encoding
proteins which are involved in vacuolar maturation and trafficking,
either directly by IL-2 or indirectly as a downstream consequence of
changes in expression of other cytokines such as IFN-, suggests that
the intracellular fate of mycobacteria might be modulated by
administration of rhuIL-2.
The observation that cytochrome oxidase type I is up-regulated in
response to IL-2 is also interesting in that it suggests that changes
in oxidative metabolism may be a feature of the increased antigen-specific activation following administration of rhuIL-2. Cytochrome oxidase is located in the mitochondrial membrane and is the
last component in the chain of electron transport resulting in
production of ATP (5). The differential expression of this mRNA species induced by rhuIL-2 administration may indicate an increased level of mitochondrial respiration associated with enhanced leukocyte activation and/or increased phagocyte oxidative metabolism in
response to increased production of IFN- by T cells.
Cytokine-induced changes in the expression of genes encoding various
oxidase components were recently described for another system. Patients
with chronic granulomatous disease treated with IFN- improved
clinically, with a reduction in the incidence of infections
(39). IFN- administration in these patients, as well as
in vitro stimulation of monocytes and neutrophils with IFN-,
resulted in a significant increase in the level of a cytosolic NADPH
oxidase mRNA. The observation that cytochrome oxidase type I is
differentially regulated in this system may be important in the control
of mycobacterial infection for another reason. Increases in cytochrome
oxidase would result in increases in ATP levels in the macrophages.
Since ATP has been shown to induce apoptosis of mycobacterium-infected
macrophages and decreased viability of intracellular mycobacteria,
up-regulation of this pathway may also be involved in the clearance of
bacilli in the patients treated with rhuIL-2 (23, 27).
The different pattern of differential regulation of genes in the peripheral blood leukocytes compared to that in the cells migrating into the site of PPD deposition in the skin is of interest. The fact that genes are not found to be differentially expressed does not prove conclusively that they are not affected by rhuIL-2 treatment. Rather, our observations suggest that these genes are not obviously modulated in PBMC in response to rhuIL-2 treatment of patients. Thus, we may have confirmation for selective effects of rhuIL-2 treatment on cells migrating to an antigen-specific response site compared to effects on cells in the circulation.
Our results indicate that differential-display technology can be used to identify subtle changes in transcriptional expression of genes in cells of tissues or blood samples obtained during rhuIL-2 (or other immunomodulatory) adjunctive therapy. Because of the very limited quantity of patient RNA available for these assays, it is not possible to expand the analysis of differential regulation of putatively identified transcripts by using the most quantitative assay, i.e., Northern blotting. We therefore used PBMC from normal PPD-positive donors to confirm that the putatively identified transcripts are indeed differentially regulated following exposure to rhuIL-2. When cells stimulated by M. tuberculosis were treated with rhuIL-2 in vitro, we observed up-regulation of the same genes, this time by utilizing a quantitative assay, i.e., Northern blotting. Thus, our studies have identified a number of novel candidate genes whose expression can now be studied in detail in order to evaluate their role in the antituberculous response which is enhanced by the administration of rhuIL-2.
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ACKNOWLEDGMENTS |
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We thank Victoria H. Freedman for help with the manuscript, Judy Adams for computer-generated graphics, and Marguerite Nulty for secretarial assistance.
This work was supported by funding from Chiron Corporation and by Public Health Service grants AI 33124, AI 40314, and AI 42056.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Cellular Physiology and Immunology, Rockefeller University, Box 176, New York, NY 10021. Phone: (212) 327-8375. Fax: (212) 327-8875. E-mail: kaplang{at}rockvax.rockefeller.edu.
Present address: Instituto Politecnico Nacional, Departmento de
Immunologia, Mexico City, Mexico.
Editor: R. E. McCallum
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Infect Immun, June 1998, p. 2426-2433, Vol. 66, No. 6
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