Download Tissue after Acute Infection Ex Vivo Cytokine Gene Expression in

HIV-1 Does Not Provoke Alteration of
Cytokine Gene Expression in Lymphoid
Tissue after Acute Infection Ex Vivo
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of May 7, 2017.
Annette Audigé, Erika Schlaepfer, Athos Bonanomi, Helene
Joller, Marlyse C. Knuchel, Markus Weber, David Nadal and
Roberto F. Speck
J Immunol 2004; 172:2687-2696; ;
doi: 10.4049/jimmunol.172.4.2687
http://www.jimmunol.org/content/172/4/2687
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References
The Journal of Immunology
HIV-1 Does Not Provoke Alteration of Cytokine Gene
Expression in Lymphoid Tissue after Acute Infection Ex Vivo1
Annette Audigé,2* Erika Schlaepfer,* Athos Bonanomi,§ Helene Joller,† Marlyse C. Knuchel,3*
Markus Weber,‡ David Nadal,§ and Roberto F. Speck2*
H
uman immunodeficiency virus severely compromises
the cellular immune system; immunomodulation offers
one promising therapeutic strategy (1– 4). Currently,
several drugs are in clinical trials based on their potential cytotrophic or antiviral actions in HIV infection. These include IL-2 (5–
8), IFN-␣ (9 –11), and GM-CSF (12).
A detailed understanding of how HIV succeeds in dysregulating
the cytokine network would be highly beneficial to the development of immune-based therapies. Some studies have linked disease
progression to a shift from a Th1 to a Th2 cytokine response (13–
15); however, these findings are still controversial (16, 17). Others
proposed a Th1 to Th0 switch or alteration of both Th1 and Th2
cytokine production without a polarized Th1/Th2 state (18, 19).
Long-term immune control of viral infections depends largely
on the cytokine milieu generated by the innate immune system in
the first 3–5 days (20). A number of in vitro studies have tried to
recapitulate the early effects of HIV infection and viral gene products on cytokines (21–23). Results depended strongly on the ex*Division of Infectious Diseases and Hospital Epidemiology, †Institute of Clinical
Immunology, and ‡Clinic of Visceral and Transplantation Surgery, University Hospital of Zurich, and §Division of Infectious Diseases, University Children’s Hospital
of Zurich, Zurich, Switzerland
Received for publication May 19, 2003. Accepted for publication December 4, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
A.A., E.S., and R.F.S. were supported by Grant 3339-64124.00 from the Swiss
National Science Foundation and grants from the OPO and the EMDO Foundations.
A.B. and D.N. were the recipients of grants from the EMDO and SWISS Bridge
Foundations.
2
Address correspondence and reprint requests to Dr. Annette Audigé or Dr. Roberto
F. Speck, Division of Infectious Diseases and Hospital Epidemiology, University
Hospital of Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland. E-mail addresses:
[email protected] and [email protected]
3
Current address: Division of Immunology and Allergy, University Hospital of Lausanne, CH-1011 Lausanne, Switzerland.
Copyright © 2004 by The American Association of Immunologists, Inc.
perimental design and were contradictory even for the same HIV
gene product. For example, Nef induced IL-2 in T cells stimulated
with CD3 and CD28 (22) but down-regulated IL-2 in T cells
treated with PHA or PMA (21). Another study reported no differences in the levels or kinetics of cytokine secretion between uninfected and acutely HIV-infected IL-2-stimulated PBMC (24).
Many reported in vitro findings are difficult to extrapolate to the
in vivo situation. They were obtained in PBMC activated with Abs
or polyclonal mitogens, monocyte-derived macrophages, or cell
lines exposed to a high virus input or recombinant HIV proteins.
However, the cytokine milieu generated in vivo after infection
results from the interaction of many different cells. Furthermore,
HIV is primarily a disease of lymphoid tissue, and thus, results
obtained in PBMC may not adequately mirror the critical immunopathogenetic events in HIV infection.
Cultures of human lymphoid tissue as blocks, also called lymphoid histocultures, offer an excellent alternative model for the
study of HIV pathogenesis (25). Histocultures preserve the rich
repertoire of cellular subpopulations and tissue architecture of
lymphoid tissue, and they support vigorous HIV replication without the prior activation needed by PBMC.
In the current work, we aimed to define the cytokine response in
lymphoid tissue after ex vivo infection with HIV. We focused on
the Th1 cytokines (IFN-␥, IL-2, and IL-12), the Th2 cytokines
(IL-4 and IL-10), the proinflammatory cytokines (IL-1␤, IL-6, and
IL-8), and TNF-␣. We also included IL-15, which shares biologic
activities with IL-2; TGF-␤, which has complex immune regulatory functions; and the ␤-chemokines RANTES and macrophageinflammatory protein (MIP)4-1␣.
4
Abbreviations used in this paper: MIP, macrophage-inflammatory protein; R5,
CCR5 tropic; X4, CXCR4 tropic; HLAC, human lymphocyte aggregate culture; DC,
dendritic cell; HMBS, hydroxymethylbilane synthase; NFIB-2, NF I; MNE, mean
normalized gene expression; SEB, staphylococcal enterotoxin B; p.i., postinfection;
PDC, plasmacytoid DC; MDC, myeloid DC.
0022-1767/04/$02.00
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The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV
infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human
lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte
aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using
distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs
for IL-1␤, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants.
Although IL-1␤, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was
similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result
in characteristic mRNA profiles for IL-1␤, -2, -4, -6, -8, -10, -12, -15, IFN-␥, TNF-␣, TGF-␤, and ␤-chemokines in tissue blocks
or HLAC. The increased expression of IL-1␤, -6, and -8 in tissue blocks may approximate clinical situations with heightened
immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may
contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern
HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV
uses during acute infection. The Journal of Immunology, 2004, 172: 2687–2696.
2688
Materials and Methods
Lymphoid tissue
The acquisition and processing of lymphoid tissue were approved by the
local ethical committee. Human tonsils from otherwise healthy patients
(18 –70 years of age) were obtained within 1–5 h after tonsillectomy from
the Department of the Ear, Nose, and Throat Surgery at University Hospital
of Zurich. The median age of the patients was 26 years. Lymph nodes of
intestinal or inguinal origin were obtained from organ donors from the
Department of Surgery, University Hospital of Zurich.
Lymphoid tissue was divided into tissue blocks of 2–3 mm3, and three
or two blocks from tonsils or lymph nodes, respectively, were placed at the
air-liquid interface of a collagen sponge gel (Pharmacia and Upjohn,
Kalamazoo, MI) covered by a membrane (pore size, 4 ␮m; Millipore,
Billerica, MA) in 1.5 ml of culture medium per well in a 12-well plate.
HLAC were prepared by transferring minced tissue and tissue fragments
into a cell strainer (70 ␮m; BD Biosciences, San Jose, CA) and grinding
the tissue through the sieve with a syringe plunger. Erythrocytes were lysed
with ACK cell lysing buffer (Cambrex, Walkersville, MD). Lymphoid cells
were washed and transferred to a 96-well plate at a concentration of 2 ⫻
106 cells/well.
Tissue blocks and HLAC were cultured in RPMI 1640 containing 15%
FCS, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 2.5 ␮g/ml fungizone,
2 mM L-glutamine, 1 mM sodium pyruvate, and 1% nonessential amino
acids.
One experiment comprised data from one donor; the numbers of experiments and replicates are given in the legends. In each single experiment,
tissue blocks and HLAC were from the same donor.
Immunostaining and flow cytometry
For the analysis of CD4⫹ and CD8⫹ T cells, cells were simultaneously
stained with mAbs (BD Biosciences) for the cell surface markers CD4 (PE;
no. 555347), CD8 (FITC; no. 555634), CD25 (Cy; no. 555433), and
CD45RA (allophycocyanin; no. 550855), or for CD4, CD8, CXCR4 (Cy;
no. 555975), and CCR5 (allophycocyanin; no. 556903); all mAbs were
used at a dilution of 1/10. Dendritic cells (DC) were quantified with a
mixture of Abs (four-color dendritic value bundle; no. 340565), NK cells
by the simultest (no. 340042), both from BD Biosciences. Analysis by flow
cytometry was performed on a FACSCalibur (BD Biosciences) and FlowJo
software (Tree Star, Ashland, OR) or CellQuest Pro software (BD Biosciences). Data for CD4⫹ and CD8⫹ T cells were derived from tonsils, and
for DC and NK cells from tonsils or lymph nodes.
Real-time quantitative PCR
RNA was extracted from lysates of samples with the RNeasy mini-kit
(Qiagen, Valencia, CA) according to the manufacturer’s instructions. For
tissue homogenates, blocks were disrupted with a mortar and pestle in lysis
buffer containing 2-ME. Cells from HLACs were lysed in lysis buffer by
vortexing and pipetting. After extraction, RNA was pooled from replicates.
To remove residual DNA, RNA preparations were treated with DNAfree
(Ambion, Austin, TX) according to the manufacturer’s instructions. RNA
quality, as judged by gel electrophoresis, was comparable in all samples;
however, the total amount of RNA from tissue blocks as determined by
spectrophotometry usually declined over time.
Reverse transcription was performed with 2 ␮g of total RNA in a total
volume of 20 ␮l. RNA was incubated with 500 ng of oligo(dT) primer
(Life Technologies, Grand Island, NY) and 500 ␮M dNTPs (Roche, Basel,
Switzerland) at 65°C for 5 min, followed by incubation on ice for 5 min.
Samples were then incubated with 3 mM MgCl2 (Promega, Madison, WI)
and 24 U of RNase inhibitor (Roche) in 1⫻ ImProm-II RT reaction buffer
(Promega) at 42°C for 2 min. ImProm-II RT (200 U; Promega) was added,
and reactions were performed at 42°C for 50 min, followed by inactivation
at 65°C for 15 min. Residual RNA was digested with 500 ng of RNase
(Roche) at 37°C for 20 min. False-positive signals from amplified residual
genomic DNA were excluded by control reactions in which ImProm-II RT
was replaced by diethylpyrocarbonate-treated-water; no signal was observed in these samples.
For real-time PCR, primers (Microsynth, Balgach, Switzerland) and
TaqMan probes (Biosearch Technologies, Novato, CA; or Applied Biosystems, Foster City, CA) were designed using Primer Express software
(Applied Biosystems), optimized, and validated (30). Hydroxymethylbilane synthase (HMBS) (GenBank no. X04217) was used as a housekeeping
gene and designed as an MGB probe (30). For PCR of IL-4, IFN-␣ and -␤,
and ␤-chemokines, commercially available primers and probes (assays-ondemand; Applied Biosystems) were used; for NFIB-2 (NF I) PCR, previously described primer and probe sequences were used (31). Real-time
quantitative TaqMan PCR with an ABI PRISM 7700 Sequence Detection
System and TaqMan Universal PCR Master Mix (Applied Biosystems)
was performed according to the manufacturer’s protocol. In each reaction,
50 ng of total RNA was amplified in a final volume of 25 ␮l. For designed
oligonucleotides, primers were used at a concentration of 300 nM and
probes at a concentration of 200 nM. The housekeeping gene was amplified
in separate reactions. Reactions were performed in duplicate (because IL-4
was expressed at very low levels in lymphoid tissue, its amplification was
done in triplicate). Thermal cycling conditions were as follows: 50°C for 2
min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C
for 1 min.
Data generated by real-time quantitative PCR were analyzed in two
steps. First, the mean normalized gene expression (MNE) for every sample
was determined using the software application Q-Gene (calculation procedure for MNE 2) (32). Second, normalized gene expression in samples
of interest, relative to controls, was assessed by calculating the ratio of
MNE(HIV infected) to MNE(mock infected).
Cytokine ELISA
Culture fluids from replicates were pooled, and cytokine concentrations
were quantified by enzyme immunoassays (IFN-␥; HyCult Biotechnology,
Uden, The Netherlands; and TNF-␣, IL-1␤, -6, -8, RANTES, and MIP-1␣;
R&D Systems, Minneapolis, MN), according to the manufacturer’s
instructions.
Exposure of tonsillar tissue blocks and HLAC to hypoxic
conditions or mitogen stimulation
The effect of hypoxia on mRNA expression of proinflammatory cytokines
was evaluated in HLAC subjected to 1% oxygen. Samples were harvested
for RNA extraction after 6, 16, 26, and 48 h. For examining the effects of
mitogens on cytokine mRNA expression, tissue blocks and HLAC were
immersed in culture medium containing either PMA (0.5 ␮g/ml) and ionomycin (0.05 ␮g/ml), staphylococcal enterotoxin B (SEB; 1 ␮g/ml), or PHA
(5 ␮g/ml) (all reagents from Sigma-Aldrich, St. Louis, MO) for 30 min at
room temperature before being cultured as described above. Mitogens were
maintained during culturing. Blocks and HLAC were harvested for RNA
extraction after 6, 24, and 72 h of stimulation.
HIV infection of human lymphoid tissue ex vivo
Each tissue block was infected by inoculation with 5–15 ␮l of viral stock
containing 1 ng of p24, which is consistent with described protocols (33).
HLAC were incubated overnight in 100 ␮l containing 5 ng of p24, washed
three times with PBS the next morning, and resuspended in 200 ␮l of fresh
medium. Culture medium (500 ␮l for blocks; 50 –100 ␮l for HLAC)
was replaced every 3– 4 days. HIV replication was followed by measuring the concentration of p24 in the culture medium with an in-house
p24 ELISA. For RNA extraction, samples were collected 1, 3, and 6
days postinfection (p.i.).
Viruses
Virus stocks were obtained by calcium phosphate-mediated transfection
(Promega) of 293T cells with pNL4-3, pYU-2 (National Institutes of
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We hypothesized that the cytokine milieu differs between infections with CCR5-tropic (R5) or CXCR4-tropic (X4) HIV strains.
Notably, R5 strains use CD4 and CCR5 to enter cells. Cells such
as memory T cells and macrophages express CCR5 and are therefore permissive to R5 strains (26). Typically, R5 strains are involved in the early infection and predominate until late-stage
AIDS. X4 strains use CD4 and CXCR4, receptors found predominantly on T cells. Emergence of X4 strains usually occurs in latestage HIV disease and is associated with an acceleration of the
immunodeficiency. The elucidation of the cytokine response to R5
and X4 strains may help to resolve their temporal appearance in
HIV (27, 28).
Although most studies of HIV pathogenesis in primary lymphoid tissue were performed with tissue blocks, human lymphocyte aggregate cultures (HLAC) have been reported to be of equivalent value as an experimental system but much easier to handle
(29). However, different culture modes may affect the initial HIV/
host interactions and subsequent cytokine responses. Therefore,
we performed our analyses in parallel in cultures of lymphoid tissue blocks and HLAC.
HIV AND CYTOKINES
The Journal of Immunology
2689
Table I. Expression of cell surface markers on tonsillar lymphocytes at baselinea
CD4⫹
CD8⫹
CD4⫹b
CD8⫹b
CD25c
CD45RAc
CCR5c
CXCR4c
CD25d
CD45RAd
CCR5d
CXCR4d
42 (35–56)
8 (3–12)
36 (14–61)
42 (13–91)
16 (9–32)
56 (29–79)
4 (1–22)
66 (32–95)
48 (14–82)
33 (14–72)
Data are given as median (minimum to maximum) (n ⫽ 12).
Percentage of positive cells within the lymphocyte population.
Percentage of positive cells of CD4⫹ T cells.
d
Percentage of positive cells of CD8⫹ T cells.
a
b
c
Health AIDS Research and Reference Reagent Program, Rockville, MD),
or p49.5 (34). Virus was harvested 48 h after transfection, filtered (0.22
␮m), and frozen at ⫺80°C. For mock infections, we harvested culture fluid
after transfection of 293T cells with a cloning vector, pCMV4, as outlined
above.
HIV p24 Ag ELISA
Treatment of tonsillar tissue blocks and HLAC with anticytokine Abs
Tissue blocks and HLAC were pretreated with neutralizing polyclonal Abs
against either IL-1␤, -6, and -8, or isotype Abs (10 ␮g/ml; R&D Systems)
for 4 h before being infected and cultured as described above. Abs were
maintained throughout the experiment; culture medium (350 ␮l for blocks;
100 ␮l for HLAC) was replaced every 3– 4 days.
Statistical analysis
⫹
⫹
Comparisons of the fractional distribution of CD25 cells of CD4 T cells
between HLAC and tissue blocks and of cytokine concentrations between
HIV- and mock-infected cultures were made using the two-tailed Wilcoxon
signed rank test. Comparisons of inhibition of HIV replication by neutralizing Abs against proinflammatory cytokines between tissue blocks and
HLAC were made with the two-tailed Mann-Whitney test.
Results
Phenotype of cells in lymphoid tissue cultured ex vivo
Composition and phenotype of cells in lymphoid tissues are critical parameters for cytokine responses and HIV replication. Immediately after receipt of the tissue (i.e., at baseline), CD4⫹ and
CD8⫹ T cells represented ⬃42 and ⬃8%, respectively, of the lymphocyte population in tonsils (Table I). Approximately 16% of
CD4⫹ T cells were positive for CCR5, and CXCR4 expression
was detected on ⬃56% of CD4⫹ T cells. CXCR4 expression on
Cytokine expression in mock-infected lymphoid tissue cultured
ex vivo
To define the cytokine responses to HIV infection, we first compared cytokine mRNA expression in mock-infected lymphoid cultures over time to that in uninfected/uncultured samples on day 0
(hereafter termed “baseline levels”). We found marked differences
in cytokine mRNA expression depending on the mode of culturing.
In tissue blocks, mRNAs for the proinflammatory cytokines IL-1␤,
-6, and -8 were strongly up-regulated over time; this increase was
already apparent after 1 day of culturing (Table II). In contrast,
expression of IL-1␤, -6, and -8 mRNAs was not substantially increased in HLAC (range of relative mRNA expression, 1.2- to
3.1-fold). IL-4 (Th2) was the only cytokine that was down-regulated over time, particularly in HLAC. Transcriptional up-regulation of other cytokines (i.e., IL-2, -10, -12, -15, IFN-␥, TNF-␣, and
TGF-␤) was less pronounced over time (relative expression, ⬍10fold), and no substantial differences were detected between tissue
blocks and HLAC. A similar pattern was observed in tissue blocks
from lymph nodes of intestinal or inguinal origin (Table III). The
Table II. Cytokine mRNA expression in mock-infected cultures from tonsilsa
Day 1
IFN-␥
IL-1␤
IL-2
IL-4
IL-6
IL-8
IL-10
IL-12A
IL-15
TGF-␤
TNF-␣
a
b
Day 3
Day 6
Blocks
HLAC
Blocks
HLAC
Blocks
HLAC
8.6 (4.6–14.1)
15.1 (12.3–67.7)
1.7 (0.9–3.5)
1.0 (0.5–1.0)
434.8 (24.2–607.1)
223.1 (60.2–336.8)
3.4 (1.8–4.3)
3.0 (1.7–3.7)
1.6 (1.1–2.7)
2.5 (1.9–3.2)
3.7 (1.0–5.5)
4.5 (0.0–16.1)
2.3 (0.3–6.7)
1.7 (0.0–4.7)
0.4 (0.0–0.5)
3.1 (0.2–5.9)
2.6 (0.1–12.9)
3.6 (0.5–4.9)
1.4 (0.1–4.2)
1.9 (0.1–6.4)
1.1 (0.9–2.3)
1.4 (0.7–2.0)
5.9 (1.7–11.5)
69.9 (33.9–146.4)
2.1 (0.8–2.9)
0.6 (0.2–1.1)
546.8 (110.5–1035.6)
292.2 (86.1–340.5)
2.2 (1.9–2.7)
2.9 (1.7–4.8)
2.9 (2.0–5.8)
2.9 (2.1–5.1)
2.8 (0.8–4.0)
3.6 (0.0–13.4)
2.7 (0.3–7.0)
2.5 (0.0–15.6)
0.1 (0.0–0.2)
2.4 (0.1–2.6)
1.9 (0.0–4.7)
2.0 (0.6–6.0)
2.9 (0.1–4.6)
7.2 (0.1–10.9)
1.8 (1.3–3.1)
1.3 (0.9–2.3)
5.7 (1.4–7.1)
42.9 (29.0–147.7)
3.6 (2.4–5.5)
1.0 (0.4–1.2)
549.3 (52.0–1568.9)
240.3 (26.0–603.2)
1.7 (0.6–2.0)
2.6 (0.7–3.3)
4.5 (2.6–5.4)
2.3 (1.9–2.6)
2.7 (0.6–3.7)
4.3 (0.0–8.9)
2.1 (0.4–6.1)
1.4 (0.0–9.7)b
0.2 (0.0–3.0)
1.3 (0.1–8.8)
1.2 (0.0–4.7)
2.8 (0.6–6.5)
4.2 (0.2–11.7)
9.6 (0.1–22.2)
2.8 (1.6–4.9)
1.0 (0.8–3.2)
Data are shown as median (minimum to maximum) relative to baseline levels from five tonsils for days 1 and 6, and from four tonsils for day 3.
One evaluation was excluded.
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A twin-site sandwich ELISA was performed essentially as described (35).
Briefly, a polyclonal Ab was adsorbed to a solid phase to capture p24 Ag
from a detergent lysate of virions. Bound p24 was visualized with an alkaline phosphatase-conjugated anti-p24 mAb and luminescent detection
system.
CD4⫹ T cells increased to ⬎95% after 1 day of culturing tissue as
either blocks or HLAC (data not shown). At baseline, ⬃36% of
CD4⫹ T cells expressed the activation marker CD25 (IL-2R␣) on
the cell surface; after 6 or 7 days of culture, a higher percentage of
CD25⫹ of CD4⫹ T cells was observed in HLAC than tissue blocks
(HLAC vs blocks: median, 39.5 vs 27.8%; p ⫽ 0.0039; n ⫽ 10),
whereas CD25 density, as based on fluorescence intensity, was
similar between HLAC and blocks (HLAC vs blocks: median, 220
vs 289.5; p ⫽ 0.6953). After grinding the tissue, percentage of DC
was 1.2–1.5% of all cells; plasmacytoid DC (PDC) and myeloid
DC (MDC) were defined by their lack of lineage markers and the
expression of HLA-DR and CD123 or CD11c, respectively. PDC
and MDC represented 45.1– 69.0% and 10.5–18.3%, respectively,
of lineage⫺ cells (n ⫽ 3). The percentage of NK cells was 0.4 –
0.7% of all cells in lymphoid tissue (n ⫽ 3).
2690
HIV AND CYTOKINES
Table III. Cytokine mRNA expression in mock-infected tissue blocks
from lymph nodesa
IFN-␥
IL-1␤
IL-2
IL-4
IL-6
IL-8
IL-10
IL-12
IL-15
TGF-␤
TNF-␣
a
Day 1
Day 3
n
1.3 (0.2–54.7)
3.4 (0.1– 4.4)
1.6 (0.3–5.2)
0.6 (0.4 – 4.7)
39.1 (2.9 –186.6)
167.0 (17.0 –3042.8)
1.0 (0.3–1.6)
1.5 (1.1–2.3)
0.9 (0.5–1.9)
1.1 (0.4 –1.9)
1.6 (0.5– 4.1)
0.9 (0.2–21.8)
4.5 (0.5–23.9)
0.9 (0.5–3.0)
1.7 (0.3–7.4)
12.6 (2.8 –123.4)
65.7 (5.9 –5237.0)
0.4 (0.2–29.3)
1.7 (0.9 –3.3)
1.1 (0.8 –11.4)
0.6 (0.4 –1.3)
1.0 (0.5–3.3)
6
3
6
5
6
6
3
6
5
5
6
Data are shown as median (minimum to maximum) relative to baseline levels.
Table IV. Cytokine production in cultures of mock-infected tonsillar
tissue blocksa
Day 1
Day 3
Day 6
IL-1␤ 0.4 (0.2– 0.6)
0.8 (0.4 –1.4)
0.6 (0.4 –1.5)
IL-6
72.7 (9.3–95.6)
293.9 (197.1– 421.6) 353.4 (201.1–581.9)
IL-8
43.0 (15.5– 64.0) 289.2 (185.1– 476.1) 545.4 (326.1–712.9)
a
Cytokine production is given as protein concentration (nanograms per milliliter)
in culture fluid; data are shown as median (minimum to maximum) from five tonsils
for days 1 and 6, and from four tonsils for day 3; blocks were cultured in duplicate
and culture fluids were pooled.
blocks. Thus, tonsillar tissue blocks and HLAC react to exogenous
stimuli by up-regulating Th1 and Th2 cytokines.
HIV replication in lymphoid tissue blocks or HLAC
Cytokine mRNA expression in HLAC exposed to hypoxic
conditions
We thought that stress from hypoxia might explain the dramatic
increases in proinflammatory cytokine mRNA levels in the tissue
blocks. To determine the effects of hypoxia, we measured cytokine
mRNA levels in HLAC exposed to hypoxic conditions for 2 days.
In general, levels of transcripts of proinflammatory cytokines were
higher in hypoxia-stimulated than in unstimulated HLAC (Fig. 1).
However, the increases were far less pronounced than those seen
in mock-infected blocks (Table II). Therefore, hypoxia alone cannot explain the marked increase of the proinflammatory cytokine
mRNAs when tissue is cultured as blocks.
Cytokine mRNA expression in tonsillar tissue blocks and HLAC
upon stimulation with polyclonal mitogens
To test whether the tissue is able to react to exogenous stimuli,
tissue blocks and HLAC were exposed to polyclonal mitogens
(Fig. 2). PMA/ionomycin stimulation resulted in strong mRNA
up-regulations of IL-2 and -4 and a severalfold increase of IFN-␥
mRNA. SEB generated a very similar pattern. PHA markedly induced IL-4 and -2 in HLAC and increased IL-4 severalfold in
To examine the effects of different coreceptors, we tested wellcharacterized HIV strains that use the coreceptors CXCR4 (NL4-3
and 134), CCR5 (49.5 and YU-2), or both (89.6). In most experiments, tonsillar blocks and HLAC supported replication of both
X4 and R5 viruses (Fig. 3). Peak levels of HIV replication were
typically higher after infection with X4 than with R5 strains. The
increase in replication for both X4 and R5 viruses was generally
slower in tissue blocks than in HLAC. Cultured blocks of lymph
nodes of intestinal and inguinal origin also supported productive
infection by NL4-3 or YU-2 (data not shown). In a minority of
experiments, tissue blocks did not support HIV replication exposed
to either R5 or X4 strains.
Cytokine mRNA expression in lymphoid tissue infected ex vivo
with HIV
We focused on early time points (i.e., 1, 3, and 6 days p.i.) with the
objective to elucidate the cytokine responses following immediate
exposure to virus, when virus spreading starts, and finally when
virus production is readily detectable but before virus-triggered
damage. Overall, cytokine mRNA expression in tonsillar tissue
blocks or HLAC with the prototype viruses NL4-3 or YU-2 was
not different from that in mock-infected samples at any time
point (Fig. 4). Similar results were obtained for the ␤-chemokine
RANTES and MIP-1␣ (Fig. 5). In lymph nodes, cytokine mRNA
levels also generally showed only minor differences between NL43-, YU-2-, and mock-infected blocks on days 1 and 3 p.i. (Table
FIGURE 1. Hypoxia moderately up-regulates proinflammatory cytokines in tonsillar HLAC. HLAC were exposed to 1% (f) or 20% (䡺) oxygen.
Samples were collected after 3, 16, 26, and 48 h of culturing. Cytokine mRNA amount is given as x-fold relative to baseline levels. For each sample,
cytokine mRNA expression was normalized to mRNA expression of the housekeeping gene HMBS. One of two experiments is shown.
Downloaded from http://www.jimmunol.org/ by guest on May 7, 2017
strong mRNA up-regulation of the proinflammatory cytokines IL1␤, -6, and -8 in tissue blocks over time was paralleled by increases in the respective protein concentrations in the culture fluids
(Table IV).
The Journal of Immunology
2691
FIGURE 2. Tonsillar tissue blocks
and HLAC react to exogenous stimuli
(PMA/ionomycin, SEB, or PHA) by
up-regulating Th1 and Th2 cytokines.
Cytokine mRNA amount after 6 (f),
24 (u), and 72 h (䡺) of stimulation is
given as x-fold relative to unstimulated
samples. For each sample, cytokine
mRNA expression was normalized to
mRNA expression of the housekeeping
gene HMBS (n ⫽ 1)
HIV replication in tonsillar tissue blocks treated with anticytokine Abs
To assess the significance of proinflammatory cytokines, the mRNAs of which were up-regulated in mock-infected blocks but not
HLAC, for HIV replication, we monitored NL4-3 replication in
tonsillar tissue blocks in the presence of Abs against either IL-1␤,
-6, or -8. This treatment resulted in substantial inhibition of NL4-3
replication; as expected, treatment of HLAC with Abs had only a
minor effect on virus replication (Fig. 7). In both tissue blocks and
HLAC, culturing with anti-cytokine Abs had no influence on cell
viability compared with addition of isotype Abs.
Discussion
FIGURE 3. Lymphoid tissue blocks and HLAC both support replication
of HIV strains with distinct coreceptor selectivity. Tissue blocks were infected ex vivo by applying 5–15 ␮l of virus stock containing 1 ng of p24
onto each block; HLAC were incubated with 5 ng of p24/2 ⫻ 106 cells.
Data are shown for a typical experiment and given as mean ⫾ SD from
duplicates for blocks and from triplicates for HLAC.
Throughout the course of infection, HIV subverts the immune system. However, the innate immune response, which largely determines the efficiency of the adaptive immune response to HIV, has
been poorly characterized. The cytokine response is a major component of the innate immune system. We investigated the cytokine
response to HIV in lymphoid tissue infected ex vivo as a model of
acute HIV infection. Our main observations were as follows: 1)
endogenous cytokine mRNA expression was substantially different
between lymphoid tissue blocks and HLAC; 2) tissue blocks and
HLAC supported replication of HIV strains equally well regardless
of coreceptor usage; 3) infection of lymphoid cultures ex vivo with
either X4 or R5 HIV strain did not result in a specific cytokine
profile; and 4) treatment of tonsillar tissue blocks but not HLAC
with neutralizing Abs against either IL-1␤, -6, or -8 markedly diminished HIV replication. Thus, the cytokine milieu after HIV
infection within an HIV-naive lymphoid microenvironment does
not show a Th1 or Th2 profile, which may reflect a hiding strategy
of the virus rather than the inability of the tissue to mount an
efficient immune response. Furthermore, different mechanisms
seem to govern HIV replication in tissue blocks and HLAC.
We first characterized the cell repertoire and the endogenous
cytokine milieu over time in mock-infected tonsillar tissue cultured ex vivo either as blocks or HLAC. Flow-cytometric analysis
demonstrated that the preparation of HLAC did not alter the percentages of CD4⫹ and CD8⫹ T cells, PDC, MDC, as well as NK
cells. Therefore, cultures of lymphoid tissue with their preserved
rich cell repertoire are very promising for the study of the innate
immune response to HIV.
To a great extent, cytokines are transcriptionally regulated and
expressed only transiently in response to stimuli (36 –38). HIV
itself is known to affect gene transcription of host cells (e.g., de
Downloaded from http://www.jimmunol.org/ by guest on May 7, 2017
V). In some cases, cytokine mRNA expression in lymphoid cultures was ⬎2-fold or ⬍0.5-fold, indicating up- or down-regulation,
respectively, of certain cytokines in cultures from some donor
tissues.
Consistent with the mRNA data, protein concentrations of the
cytokines IFN-␥, TNF-␣, IL-1␤, -6, and -8, and of the ␤-chemokines RANTES and MIP-1␣ in HIV-infected cultures from tonsillar tissue blocks were not significantly different from those in
mock-infected cultures (Fig. 6).
2692
HIV AND CYTOKINES
novo methylation of the IFN-␥ promoter and subsequent downregulation of IFN-␥ production) (39). Therefore, the innate immune response to HIV and the effects of the virus on cytokines are
directly reflected by the transcriptional level of cytokines. Consequently, we examined the cytokine milieu primarily by quantitative real-time PCR. Changes in mRNA expression were also verified at the protein level. We found that the culture mode
significantly influenced the constitutive cytokine expression. In
mock-infected lymphoid tissue blocks of tonsillar, inguinal, or intestinal origin, IL-1␤, -6, and -8 mRNAs were strongly up-regulated over time. In contrast, IL-1␤, - 6, and -8 mRNAs showed no
mRNA up-regulations in HLAC. The other cytokines analyzed
showed less pronounced mRNA changes whether tissue was cultured as tissue blocks or HLAC. Cytokines at the protein level
paralleled the changes in mRNA with significant increased concentrations of IL-1␤, -6, and -8 similar to reported findings (40).
No increase was observed for the other cytokines analyzed.
FIGURE 5. HIV does not provoke alteration
of chemokine gene expression in lymphoid tissue
after acute infection ex vivo. Cytokine mRNA
amounts in NL4-3-infected (A, f; B, f, Œ, 䉬)
and YU-2-infected (A, 䡺; B, 䡺, ‚, 䉫) tonsillar
cultures are given as x-fold relative to mock-infected samples. For each sample, cytokine mRNA
expression was normalized to mRNA expression
of the housekeeping gene HMBS. In A, data are
given as median (maximum-minimum) from five
experiments (from four for day 3 p.i.).
Exogenous addition of IL-1␤, -6, and -8 is known to enhance HIV
replication in in vitro models (41– 43). Therefore, we expected a more
vigorous HIV replication in tissue blocks than in HLAC. A direct
comparison of HIV replication between these two culture modes is
problematic, because cell number and viability in tissue blocks cannot
be controlled over time. Furthermore, the infection protocols are different. In any case, both culture systems showed vigorous HIV replication after infection. Along with distinct cytokine profiles, the similar HIV replication efficiencies suggest that different molecular
mechanisms likely govern HIV replication in HLAC and tissue
blocks. Notably, R5 and X4 strains replicated equally well in both
culture systems, albeit they are known to target distinct cell subsets.
We hypothesized that infection of lymphoid tissue blocks and
HLAC ex vivo with HIV would result in characteristic cytokine responses, especially that cytokine responses would differ between HIV
strains with selective coreceptor usage. However, we observed no
consistent changes in mRNA levels for proinflammatory, Th1, or Th2
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FIGURE 4. HIV does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo. Cytokine mRNA amounts in
NL4-3-infected (f) and YU-2-infected (䡺) tonsillar cultures are given as x-fold relative to mock-infected samples. For each sample, cytokine mRNA
expression was normalized to mRNA expression of the housekeeping gene HMBS. Data are given as median (maximum-minimum) from five experiments
(from four for day 3 p.i.).
The Journal of Immunology
2693
Table V. Cytokine mRNA expression in NL4-3- and YU-2-infected relative to mock-infected tissue blocks from lymph nodesa
Day 1
IFN-␥
IL-1␤
IL-2
IL-4
IL-6
IL-8
IL-10
IL-12
IL-15
TGF-␤
TNF-␣
a
Day 3
NL4-3
YU-2
NL4-3
YU-2
n
1.7 (1.0–2.9)
1.8 (0.9–8.7)
1.6 (0.8–3.6)
0.8 (0.6–7.2)
1.4 (0.7–12.1)
1.7 (1.0–3.4)
1.2 (1.2–5.4)
1.5 (1.2–2.3)
1.8 (1.6–3.3)
1.3 (0.9–1.9)
1.3 (0.6–1.6)
1.3 (1.0–2.0)
1.6 (1.2–6.7)
1.1 (0.9–1.8)
1.6 (0.8–3.5)
1.6 (1.0–2.6)
1.9 (1.1–3.3)
1.8 (1.3–3.8)
1.2 (1.1–1.9)
1.8 (1.0–4.3)
0.9 (0.7–1.1)
1.1 (1.0–1.7)
1.3 (0.1–4.8)
0.8 (0.0–3.2)
1.1 (0.5–2.9)
0.8 (0.1–3.0)
1.4 (0.7–25.3)
1.2 (0.6–12.5)
0.5 (0.0–4.3)
0.9 (0.7–3.1)
0.9 (0.1–3.7)
1.0 (0.8–1.2)
1.2 (0.3–1.8)
0.9 (0.3–1.1)
0.5 (1.2–6.7)
1.2 (0.7–2.5)
1.5 (0.3–3.4)
0.9 (0.3–7.0)
0.9 (0.1–1.9)
0.6 (0.0–1.1)
1.0 (0.4–1.5)
0.9 (0.0–2.2)
1.0 (0.9–1.4)
1.0 (0.4–1.9)
6
3
6
5
6
6
3
6
5
5
6
Data are shown as median (minimum to maximum).
cytokines are specifically produced by cell subsets, such as IL-2
by T cells (47, 48), IFN-␥ by T cells, NK cells, or macrophages
(49, 50).
Importantly, the similar cytokine patterns after challenge with
R5 and X4 HIV strains argue against a specific role for the examined cytokines in the distinct temporal appearance of viral strains.
This is all the more surprising, because R5 and X4 viruses target
distinct cell types with definite abilities to produce cytokines.
In findings similar to ours, Kinter et al. (24) did not observe
significant differences in the levels or kinetics of cytokine secretion
between uninfected and HIV-infected samples with IL-2-stimulated PBMC. Margolis et al. (51), who also examined the ␤-chemokine response to HIV in lymphoid tissue, found no differences
of MIP-1␣, -␤, and RANTES at earlier time points after infection
with a X4 strain but found a substantial increase after 8 days; no
change at all was noted after infection with a R5 strain. These and
our studies are distinguished from most other studies in that the
FIGURE 6. HIV does not alter cytokine or chemokine production in lymphoid tissue after acute infection ex vivo. Cytokine or chemokine protein
concentrations in NL4-3-infected (f), YU-2-infected (u), and mock-infected (䡺) cultures of tonsillar tissue blocks are given as median (maximumminimum) from five experiments (from four for day 3 p.i.).
Downloaded from http://www.jimmunol.org/ by guest on May 7, 2017
cytokines or ␤-chemokines. Parallel changes at the protein level excluded a major effect of HIV on posttranscriptional or translational
modulation of gene expression. This was particularly surprising, because we had selected the cytokines based on their reported changes
during HIV infection or their immunopathogenic relevance for the
innate immune response. No consistent and marked alterations were
found either, when we expanded our analyses to IFN type I (data not
shown), which is induced in various in vitro acute HIV infection models (44 – 46). Furthermore, mRNA levels of cytokines at very early
time points after infection (i.e., 6 and 16 h after HIV infection) were
also similar for HIV-infected and mock-infected samples (data not
shown).
Because we analyzed mRNA from whole-tissue lysates, distinct
cytokine responses within subsets of cells may have been missed.
However, it seems rather unlikely that selective expression of cytokines within subsets of cells will be balanced so that no difference or pattern was observed. Furthermore, some of the analyzed
2694
cytokine profiles were examined in the context of spreading HIV
infection of PBMC or lymphoid tissue. In most other studies, specific cytokine profiles were obtained using recombinant HIV proteins (52), an extremely high multiplicity of infection (53), or
monocyte-derived macrophages (54). For example, expression of
Nef in monocyte-derived macrophages results in the release of
MIP-1␣ and MIP-1␤ that has been interpreted as critical for attracting CD4⫹ T lymphocytes for subsequent HIV dissemination
(23). The work by Margolis et al. and our laboratory do not corroborate these findings. Indeed, macrophages in lymphoid tissue
cultures are selectively infected by R5 strains (55). Therefore, we
would expect that infection of lymphoid tissue with R5 strains
should recapitulate the reported increase of ␤-chemokine release.
However, exactly the opposite was true with increased ␤-chemokine release after infection with X4 strains (51), which notably do
not productively infect macrophages.
In vivo studies of patients with primary HIV infection reported
some increases, mainly for proinflammatory cytokines, but without
a marked and characteristic profile similar to the results we observed (16, 56, 57). Strikingly, patients with acute mononucleosis
(EBV) showed higher levels of cytokines in the serum than patients with primary HIV infection, suggesting that an absent or
weak cytokine response is specific to HIV infection (57). Only a
very small number of patients with acute HIV infection have been
studied for their cytokine profiles, because most patients are not
aware of acute HIV transmission and do not experience symptomatic infection. Thus, the cytokine responses in patients with asymptomatic primary HIV infection are unknown. The data we present
are consistent with moderate and not acute cytokine responses described in patients with primary HIV infection.
The critical role of the experimental system in investigating the
cytokine response to HIV is illustrated by the following facts. In
the CD4⫹ lymphoblastoid T cell line CEM 72 h after infection,
HIV infection resulted in a ⬎500-fold up-regulation of mRNA
expression of the NFIB-2 (31). In tonsillar tissue blocks, HIV infection did not result in up-regulation of NFIB-2 mRNA (data not
shown). These opposing findings emphasize the importance of primary cultures for HIV research.
The lack of a distinct cytokine response to HIV may be explained by the lymphoid tissue cultured ex vivo being anergic. This
is certainly not the case, because selective cytokine responses were
seen after exposing the tissue to polyclonal mitogens. This suggests that the dominance of IL-1␤, -6, or -8 in tissue blocks will
not override specific cytokine responses and that the lack of cytokine responses we observed is real.
Hypoxic stress or surgery may partially trigger the increased
generation of IL-1␤, - 6, and -8 in tissue blocks. Although these
cytokine increases may represent an in vitro artifact, they may
approximate clinical situations of heightened immune activation.
We made use of this phenomenon for dissecting the roles of IL-1␤,
-6, and -8 for HIV replication. In tissue blocks, neutralization of
individual cytokines resulted in substantial inhibition of HIV replication, whereas in HLAC, neutralization showed only minor effects. Similar to our findings, neutralization of TNF-␣, IL-1␤, or
IL-6 resulted in decreased HIV replication in IL-2-stimulated
PBMC (24). These findings indicate the requirement for synergistic activity of multiple cytokines for efficient HIV replication in
certain situations. Consistent with our observations, the combined
administration of TNF-␣, IL-2, IL-6, and TNF-␣ is required for
induction of HIV in latently infected CD4⫹ T cells (58).
In HLAC, an activated cellular phenotype of CD4⫹ T cells, as
illustrated by higher expression of the IL-2R␣ (CD25), may compensate for the lack of increased IL-1␤, -6, and -8 in the observed
vigorous HIV replication. HLAC also reveal an increased proportion of T cells expressing the activation marker CD95.5
In summary, although both blocks and HLAC of lymphoid tissue cultured ex vivo support HIV replication, they differ in their
endogenous cytokine profiles. Irrespective of the mode of tissue
culturing, we found no selective changes in cytokines after HIV
infection, suggesting that the initial infection process does not provoke a cytokine response. This, in turn, may justify the proposal
that, during early infection of an HIV-naive host, the virus uses
prevention of effective cytokine responses as a mechanism to escape host defenses and rapidly establish progeny. The data we
present are consistent with moderate and not acute cytokine responses described in patients with primary HIV infection.
Acknowledgments
We acknowledge Dr. S. Rampini for critically reviewing the manuscript,
and Dr. B. von Beust for technical advice. Furthermore, we thank the
Departments of Ear, Nose, and Throat Surgery, and Pathology at the University Hospital of Zurich for generous assistance in obtaining posttonsillectomy samples.
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