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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4752-4760
Individual Cell Analysis of the Cytokine Repertoire in Human
Immunodeficiency Virus-1-Infected Monocytes/Macrophages by a
Combination of Immunocytochemistry and In Situ Hybridization
By
Ruth Esser,
Wolfgang Glienke,
Reinhard Andreesen,
Ronald E. Unger,
Marina Kreutz,
Helga Rübsamen-Waigmann, and
Hagen von Briesen
From Georg-Speyer-Haus, Frankfurt am Main, Germany; the Department of
Hematology and Oncology, University of Regensburg, Regensburg, Germany;
the Neurological Institute (Edinger Institute), University of
Frankfurt/Main, Germany; and Bayer AG, Pharma Research Center,
Wuppertal, Germany.
 |
ABSTRACT |
The expression of many cytokines is dysregulated in individuals
infected with the human immunodeficiency virus-1 (HIV-1). To determine
the effects of HIV-1 infection on cytokine expression in individual
cells (at the single cell level), we investigated the intracellular
levels of proinflammatory cytokines (tumor necrosis factor [TNF]- ,
interleukin [IL]-1 , IL-6, and IL-8) and hematopoietic growth
factors (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF]) in monocyte-derived macrophages, mock-infected, or infected with HIV-1 by
immunocytochemical staining for cytokine protein and compared this with
secreted cytokine levels as determined by specific enzyme-linked
immunosorbent assay (ELISA). No difference in the frequency or
intensity of cell-associated immunocytochemical cytokine staining could
be observed between HIV-1 and mock-infected cells even though the level
of secreted proinflammatory cytokines increased and the hematopoietic
growth factors decreased in HIV-1-infected cultures. Furthermore,
equal expression of cytokine mRNA was observed in all cells in the
culture regardless of whether the cells were productively infected with
HIV-1 as determined by double-labelling immunocytochemical staining for
HIV-1 p24 antigen and in situ hybridization for cytokine mRNA
expression. These results indicate that HIV-1 infection results in
dysregulation of intracellular cytokine mRNA expression and cytokine
secretion not only in HIV-1-infected cells, but also through an
indirect way(s) affecting cells not producing virus.
| |
INTRODUCTION |
INFECTION BY THE HUMAN immunodeficiency
virus-1 (HIV-1) initiates a slowly progressing degenerative disease of
the immune system, termed the acquired immunodeficiency syndrome
(AIDS). Beside lymphocytes, cells of the macrophage lineage are major target cells for HIV-1.1-3 HIV-1-infected macrophages
persist in tissues for extended periods of time containing latent
proviral DNA or large numbers of infectious particles within
cytoplasmic vacuoles.4 Furthermore, monocytes/macrophages
(MO/MAC) may be important as vehicles for viral dissemination
throughout the body. In tissues such as the lung and the brain, HIV-1
is located primarily in macrophage-like cells (ie, alveolar macrophages
and microglia, respectively).5,6 Macrophages are also
believed to be a vehicle for the transmission of the virus between
individuals because for mucosal infection, it was found that a crucial
property of the transmitted HIV-1 variant is its tropism for
macrophages.7
Macrophages are major effector cells of the immune system and play an
essential role as regulator cells in hematopoiesis. Many of the
immunoregulatory and effector functions are mediated by cytokines
secreted by macrophages under a variety of physiologic and
pathophysiologic conditions. Disturbances in the production of
cytokines in macrophages and other immune cells by HIV-1 infection may
bring about immune dysfunction, which leads to AIDS.4,8,9 In addition, abarrant cytokine secretion may lead to a cascade of
secondary events that are likely to cause the wasting syndrome, neurologic manifestations of disease, and changes in T-cell responses (ie, switching from a T helper 1 to a T helper type 2 activity).10-12
Cytokines influence the activity of the immune system and their
regulation is important in maintaining an effective immune response.13 Interleukin (IL)-1, IL-6, and tumor necrosis
factor (TNF)- are cytokines involved in the regulation of
inflammation.14,15 IL-8 is an activating factor for
neutrophils with chemotactic activity for migrating immune
cells.16,17 The hematopoietic growth factors, granulocyte
colony-stimulating factor (G-CSF) and granulocyte-macrophage
colony-stimulating factor (GM-CSF), regulate the proliferation and
differentiation of hematopoietic progenitor cells.18,19 In
addition to their proliferative role, they contribute to maintaining
cell viability and stimulate the functions of mature macrophages and
granulocytic cells.20
Dysregulation of cytokine production by macrophages infected with HIV-1
can be demonstrated in vitro. Increased secretion of proinflammatory
cytokines IL-1, IL-6, IL-8, and TNF- has been observed in
HIV-1-infected macrophages.8,9,21,22 On the other hand, a
downregulation of the hematopoietic growth factors M-CSF, G-CSF, and
GM-CSF was observed when macrophages were infected with
HIV-1.23
The goal of this study was to examine if the dysregulated cytokine
secretion of MO/MAC due to HIV-1 infection can be correlated to an
altered pattern of cytokine expression in these cultures at the
single-cell level. Immunocytochemical staining for cellular cytokine
protein expression and an enzyme-linked immunosorbent assay (ELISA) for
secreted cytokine measurement was used to examine the pattern of
cellular cytokine protein expression in individual cells compared with
secreted cytokine levels. Furthermore, a double-labelling method
combining immunocytochemistry for HIV-1 p24 antigen detection and in
situ hybridization for cytokine mRNA was used to simultaneously examine
individual cells for the effects on the expression of cytokine mRNA by
HIV-1 replication within these cells.
| |
MATERIALS AND METHODS |
Isolation and culture of peripheral blood mononuclear cells.
Peripheral blood mononuclear cells (PBMC) were isolated from healthy
donors by density gradient centrifugation and cultured in supplemented
RPMI 1640 with 5% heat-inactivated human AB serum on hydrophobic
Teflon foils.24 The MO-derived MAC were separated by
adherence in plastic chamber slides (Nunc, Wiesbaden, Germany) and
cultured in RPMI 1640 with 5% human AB serum. Cells were fixed for
immunocytochemistry with 4% paraformaldeyde at the indicated time
points. The adherent cell layer (6 to 10 × 105
MAC/chamber slide) consisted of up to 95% MAC as judged by morphology, nonspecific esterase staining, and expression of CD14 antigen. Detection of CD14 was performed by immunocytochemistry using the monoclonal antibody (MoAb) My 4 (Coulter, Hamburg, Germany).
HIV-1.
Preparations of HIV-1 stock were obtained by propagating the virus in
peripheral blood T cells or MAC cultures, respectively, and harvesting
the culture at the peak of infectivity. The cell suspension
(106 cells/mL) and cell-free supernatant with a reverse
transcriptase activity of 5 × 105 to 1 × 106 cpm/mL/90 minutes was stored in aliquots
at  70°C until further use.3 Only mycoplasma-free
virus stocks, tested with a mycoplasma tissue culture DNA probe assay
(Gen-Probe, San Diego, CA), were used. We used the monocytotropic
strain HIV-1D117III derived from a perinatally infected
child. It rapidly replicates to a high titer in MAC.3,25 As
a control, cell suspension and cell-free supernatant were prepared from
uninfected cultures corresponding the protocol for stock virus and used
for mock infection.23
HIV-1 infection of MAC.
The PBMC were infected with 1 mL stock virus per 10 mL cell suspension
in Teflon bags. Cultures were inoculated either with HIV-1-infected
T-cell suspension on day 1 (protocol 1) or cell-free supernatant of
HIV-1 MAC cultures on day 8 (protocol 2). Control cultures were
incubated with corresponding mock material (see above). After 7 days
postinfection, MO/MAC were separated by adherence whereby the virus
inoculum and the nonadherent cells were removed by washing the cell
layer several times with serum-free medium. The infection of the MO/MAC
cultures was demonstrated by determination of HIV-1 antigen
concentration in the supernatant of the cultures using an HIV-1 antigen
ELISA (Organon Teknika, Eppelheim, Germany).
Stimulation of MAC and determination of cytokine secretion.
At the indicated time points, MAC were stimulated for 4 and 24 hours in
fresh medium with or without 100 ng/mL lipopolysaccharides (LPS,
Salmonella abortus equi, kindly provided by C. Galanos, Max
Planck Institut, Freiburg, Germany). Cell supernatant was harvested,
filtered through 0.22 µm membranes (Millipore, Eschborn, Germany),
aliquoted, and stored at 70°C. The supernatants were analyzed using specific ELISAs for the following cytokines: IL-1, IL-6, IL-8, G-CSF, GM-CSF (R & D Systems, Minneapolis, MN); TNF- (Endogen, Boston, MA). The amount of cytokine measured with ELISA was
normalized to the cell number counted in the well at the time when the
supernatant was harvested.
Immunostaining for cytokines.
After cultivation, the cells were washed with phosphate-buffered saline
(PBS), fixed with 4% paraformaldehyde in PBS, and stored in 70%
ethanol at 20°C until use. The cells were incubated for 30 minutes with 0.1% saponin, which permeabilized the cell membranes for
antibody interaction with intracellular antigens. After washing with
PBS, the cells were exposed to 10% human serum for 30 minutes followed
by incubation with one of the monoclonal antibodies overnight at
4°C: mouse antihuman IL-1 (1:300, FIB-3, Dianova,
Hamburg, Germany); mouse antihuman IL-6 (1:10, IL-6-8, Boehringer
Mannheim, Mannheim, Germany); mouse antihuman TNF- (1:1,000, TNF-E,
kindly provided by G.R. Adolf, Bender GmbH, Vienna, Austria), mouse
antihuman IL-8 (1:1,000, subclone 4G9/A5/A7, kindly provided by M. Ceska, Sandoz, Vienna, Austria), mouse antihuman G-CSF (1:100, clone
5.24, Oncogene Science, Uniondale, NY), mouse antihuman GM-CSF (1:100,
code ZM213, Genzyme, Cambridge, MA). As a negative control (1:20,
isotype control) IgG1 was used (Dianova). Afterwards the cells were
exposed to peroxidase-conjugated goat antimouse immunoglobulins (1:100,
Dako, Hamburg, Germany) for 30 minutes, peroxidase-conjugated rabbit
antigoat immunoglobulins (1:100, Dako) for 30 minutes, and finally the
peroxidase reaction was developed by incubation with diaminobenzidin
(DAB-kit Vectastain; Vector Laboratories, Burlingame, CA), which
resulted in a brown reaction product. For quantitation, positive and
negative cells were counted using a 25X objective. A total of 200 cells
were counted two times for each slide and the mean was calculated. This
evaluation was performed exemplarily by two individuals and no
considerable difference was found. The statistical significance was
calculated using the  2 test.
Double-labelling methodology: HIV-1 p24 immunostaining and in situ
hybridization for cytokine mRNA detection.
After cultivation, the cells were washed with PBS, fixed with 4%
paraformaldehyde in PBS, and stored in 70% ethanol at 20°C until use. The peroxidase immunostaining with Vectastain Elite ABC-System (Vector Laboratories) was performed according to the manufacturer's protocol. Briefly, the fixed cells were rehydrated in
PBS for 15 minutes and exposed to normal human serum for 30 minutes,
HIV-1 anti-p24 antibody (mouse MoAb Kal-1, 1:100, Laboserv, Eggenstein,
Germany) overnight at 4°C and biotinylated antimouse antibody for
30 minutes. Incubation with ABC-Elite reagent containing avidin and
biotinylated horseradish peroxidase was performed for 30 minutes at
room temperature. Incubation with peroxidase substrate 3-amino-9-ethylcarbazole (AEC) was performed until color developed. The
slides were then incubated with 2 × sodium sodium citrate (SSC)
at 60°C for 10 minutes to prepare them for in situ hybridization. Prehybridization for 1 hour at 37°C was followed by hybridization overnight at 37°C with 200 ng/mL antisense oligoprobe labeled with
digoxigenin (IL-8 BPR 100; IL-6 BPR 32; TNF- BPR 49, British Biotechnology, Oxon, UK). Washing conditions were: 2 × 10 minutes with 4 × SSC/30% formamide at 37°C, 2 × 10 minutes with 2 × SSC/30% formamide at 37°C and 0.2 × SSC/30% formamide at 37°C, as well as 2 × 10 minutes washing
with Tris/HCl/bovine serum albumin (TBS)/0.1% Triton X-100. After the
washing steps, 1:600 diluted antidigoxigenin antibody (Boehringer
Mannheim) was added for 60 minutes at 37°C. After 2 × 10 minutes washing with TBS, the samples were incubated with revealing
buffer (100 mmol/L TrisHCl pH 9.5, 100 mmol/L NaCl, 5 mmol/L
MgCl2; 0.2 mmol/L 5-bromo-4-chloro-3-indolyl phosphate [BCIP], 0.2 mmol/L nitroblue tetrazolium salt [NBT]) for 3 hours at
37°C.
| |
RESULTS |
Immunocytochemical identification and secretion of proinflammatory
cytokines in uninfected MO/MAC.
Intracellular protein localization and secretion of proinflammatory
cytokines (TNF-, IL-1, IL-6, IL-8) were examined in unstimulated
and LPS-stimulated MO/MAC cultures on day 8 and day 15 after start of
culture. Intracellular cytokines were demonstrated using
immunocytochemical techniques and specific MoAbs. In unstimulated MAC,
no intracellular IL-1, IL-6, and TNF- were detected. In contrast,
a constitutive expression of IL-8 protein was demonstrated by a
positive staining in about 75% to 90% of the cells
(Table 1). After
LPS-stimulation IL-1, IL-6, and TNF- were produced and the
staining for IL-8 became more intense in 41% to 56% of the positive
cells (Table 1). A different pattern of staining during stimulation
with LPS for the cytokines IL-6, IL-8, and TNF- occurred compared
with IL-1. After 4 hours of stimulation with LPS, MAC exhibited a
granulated staining in distinct parts of the cytoplasma for IL-8
(Fig 1B), as well as for IL-6 and TNF- (data not shown). After 24 hours, a diffuse staining of the whole cell
was observed, as shown for IL-8 in Fig 1C. IL-1 producing MAC on the
other hand, exhibited diffuse cytoplasmic staining both after 4 hours
and 24 hours (Fig 1D and E). Positively stained cells were counted and
the results are summarized in Table 1. IL-1 was detectable after 4 hours of stimulation in the majority of the cells (64% to 85%). After
24 hours, a more intense staining for IL-1 was observed in 5% to
10% of these cells. TNF- production was detectable after
stimulation with LPS for 4 hours in 7% to 14% of the cells clearly
identified by the characteristic granulated staining. After 24 hours,
the percentage of TNF--synthesizing cells increased to 43% to 61%
with a diffuse staining. A total of 10% to 15% of the cells produced
IL-6 after 4 hours of stimulation and 50% to 80% after 24 hours.
Staining with IL-8 antibody resulted in 54% to 96% positive cells in
unstimulated and LPS-stimulated cultures. The response of the cells to
LPS was reflected in the additional occurrence of characteristic
granular staining after 4 hours of stimulation and subsequently in a
strong diffuse staining after 24 hours. The secretion of cytokines in
the corresponding supernatant was assayed with the ELISA technique to
compare with the immunocytochemical expression of cell-associated
cytokines. Without stimulation, MAC did not release IL-1, IL-6, and
TNF-, whereas for IL-8, a constitutive secretion was observed
(Table 2). The amount of IL-8 that was
secreted, however, was strongly enhanced by stimulation with LPS.
Corresponding to the enhancement in the percentage or intensity of
cellular staining after 24 hours of stimulation, the secretion of
IL-1, IL-6, and IL-8 increased further between 4 hours and 24 hours,
whereas TNF was maximally secreted after 4 hours of stimulation.
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Table 1.
Immunocytochemical Evaluation of Intracellular
Proinflammatory Cytokines and Hematopoietic Growth Factors in
Uninfected and HIV-1-Infected Macrophages
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| Fig 1.
Immunocytochemical staining of uninfected MAC. By using
IgG1 antibodies for negative control (isotype control), only an
unspecific staining of the area of the nucleus of the cells was seen
(A). With specific anti-IL-8 antibodies, intracellular IL-8 could be detected after 4 hours stimulation with LPS as granulated staining (B)
and after 24 hours LPS as diffuse staining (C). After 4 hours stimulation with LPS, low amounts of IL-1 could be detected (D) and
after 24 hours LPS, a strong specific staining was observed (E).
Intracellular G-CSF could be detected in unstimulated cultures (F), as
well as in LPS-stimulated cultures (G) (× 1,500).
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Table 2.
Secretion of Proinflammatory Cytokines and Hematopoietic
Growth Factors by Uninfected and HIV-1-Infected Macrophages
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Immunocytochemical identification and secretion of hematopoietic
growth factors in MO/MAC.
Cell-associated cytokines and secretion of the hematopoietic growth
factors G-CSF and GM-CSF was examined in unstimulated and
LPS-stimulated MO/MAC cultures. Intracellular G-CSF could be detected
in 68% to 98% of the cells in unstimulated and LPS-stimulated MAC
cultures (Table 1, Fig 1F and G). The percentage of GM-CSF-producing cells in unstimulated as well as LPS-stimulated cultures was 82% to
99% (Table 1). Secretion of both G-CSF and GM-CSF was not detected in
the supernatant of unstimulated MO/MAC cultures until the cultures were
stimulated with LPS (Table 2).
Immunocytochemical identification and secretion of proinflammatory
cytokines and hematopoietic growth factors after infection with HIV-1.
It has been reported that the secretion of proinflammatory cytokines
and hematopoietic growth factors are differentially regulated after
infection with HIV-1: an increased secretion of proinflammatory
cytokines and a decreased secretion of hematopoietic growth factors has
been shown in HIV-1-infected cultures compared with uninfected
cultures from the same blood donor.8,9,21-23 To further
examine this differential regulation at the single cell level by
immunocytochemical staining, we analzyed the expression of
proinflammatory cytokines and hematopoietic growth factors in
individual cells after infection of the culture with HIV-1. The
frequency of cytokine positive cells and the intensity of staining for
both the proinflammatory cytokines as well as the hematopoietic growth
factors, was identical in HIV-1-infected and uninfected cultures
(Table 1). However, the levels of the secreted proinflammatory
cytokines and hematopoietic growth factors measured in the
corresponding supernatants of these cultures showed a twofold to
fivefold higher level of proinflammatory cytokines in the infected
cultures compared with the uninfected control cultures (Table 2),
whereas G-CSF and GM-CSF secretion was reduced on infection by a factor
of 2 to 6 (Table 2). To exclude that these effects may be due to
cytokines in virus inoculum, we measured cytokine levels (ie, IL-1,
IL-6, IL-8, TNF-, G-CSF, GM-CSF, and M-CSF) in the virus and the
mock inoculum. We found nearly identical cytokine amounts in the virus
inoculum and the mock inoculum (data not shown).
Detection of HIV-1 p24-positive cells and cytokine mRNA expressing
cells by combined immunocytochemistry and in situ hybridization.
To determine whether HIV-1-infected cells, in particular, were
responsible for the increased secretion of proinflammatory cytokines or
whether an indirect effect was responsible for the changes in cytokine
secretion, a double-labelling methodology was used to examine both the
cytokine mRNA expression and HIV-1 infection in the same cell.
Cell-associated cytokine mRNA for IL-8, IL-6, and TNF- was detected
by in situ hybridization and HIV-1 p24 antigen by immunocytochemistry.
After stimulation with LPS for 24 hours, the same percentage of
cytokine mRNA positive cells was found in HIV-1-infected and
uninfected cultures: 78% to 86% MAC were IL-8 mRNA positive, 58% to
68% were IL-6 mRNA positive, and 41% to 50% were TNF- mRNA
positive (Table
3). Figure 2B shows the three possible outcomes of
immunocytochemical staining for HIV-1 p24 antigen and in situ
hybridization for cytokine mRNA expression. Cell no. 1 is both
p24-negative and IL-8 mRNA negative, cell no. 2 exhibits a positive
signal for IL-8 mRNA, but no p24 antigen expression, and cell no. 3 exhibits both IL-8 mRNA and p24 expression. In the HIV-1-infected
cultures, the percentage of cytokine-mRNA positive cells was similar
either in the proportion of the p24-positive, as well as p24-negative
MAC (Table 3).
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Table 3.
Detection of HIV-1 p24-Positive Cells and Cytokine
mRNA-Expressing Cells by Combined Immunocytochemistry and In Situ
Hybridization
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| Fig 2.
Detection of HIV-1 p24-positive cells and cytokine mRNA
expressing cells by combined immunocytochemistry and in situ
hybridization. (A) Negative control (LPS-stimulated, uninfected MAC
hybridized with IL-8 sense oligonucleotides). (B) Simultaneous
detection of HIV-1 p24 (red staining) with immunocytochemistry and IL-8 mRNA in LPS-stimulated MAC with in situ hybridization (dark granules) at day 22 after start of culture. Cell no. 1, HIV p24-negative and
IL-8-negative; cell no. 2, HIV p24-negative and IL-8-positive; cell
no. 3, HIV p24-positive and IL-8-positive (×1,500).
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DISCUSSION |
Despite the existence of conflicting data, most results show a
dysregulated cytokine production in HIV-1 infection. Elevated levels of
proinflammatory cytokines such as IL-1, IL-6, IL-8, and TNF- have
been found in the sera of HIV-1-infected individuals.26-29 In addition, cells from HIV-1-infected patients cultured in
vitro,26,30,31-38 as well as in vitro infected
MO/MAC,39-42 release high levels of these cytokines. The
information about the regulation of hematopoietic growth factors is
more limited. Some investigators have shown lower levels in
HIV-1-infected patients for GM-CSF,43,44 whereas others,45 have found normal or even higher levels. The
dysregulated expression of cytokines during infection with HIV-1 may
contribute to the pathogenesis of AIDS.10-12 To determine
the effects of HIV infection on the regulation of specific cytokines by
MO/MAC, we analyzed the secretion by MO/MAC of these cytokines in the
supernatant by ELISA and the expression of these cytokines at the
single cell level using an indirect immunoperoxidase staining method
and in situ hybridization.
In the absence of LPS, no secretion of IL-1, IL-6, or TNF- was
detected in either infected or uninfected MAC and intracellular staining for these proteins could not be detected by immunostaining. Similar results have been found by Molina et al46 for the
secretion of IL-1, IL-6, and TNF- and by Gan et al47
for IL-6. In contrast, other investigators found an induction of IL-6
when these cells are infected by HIV-1.30,41 This
contradiction may be due to contaminating endotoxins present in certain
sera used in culture media. Endotoxins are known to be activators of
MO/MACs.48 IL-8, on the other hand, was produced
constitutively in uninfected cells and secretion was elevated by HIV-1
infection. Constitutive expression of IL-8 in uninfected MO/MAC has
been reported by others.49 Secreted IL-1, IL-6, IL-8,
and TNF- were detected after MO/MAC were stimulated by LPS and
levels were significantly higher in HIV-1-infected cultures compared
with uninfected cultures, whereas secreted levels of G- and GM-CSF
decreased in HIV-1-infected cultures. Molina et al50
reported similar results for THP-1 cell line that acutely infected
cells compared with chronically or uninfected cells expressed
significantly higher levels of LPS-induced IL-1, IL-6, and TNF-
on protein and at the mRNA level. These results indicate that HIV-1
potentiates the inflammatory response of MO/MAC and suppress
hematopoietic activities.
LPS stimulation of uninfected MO/MAC resulted in the induction of
intracellular IL-1, IL-6, and TNF- and IL-8 staining became more
intense (Fig 1 and Table 1). A characteristic, granulated staining in
juxtanuclear position for TNF-, IL-6, and IL-8 early after LPS
stimulation was observed. Others51,52 observed a similar
staining pattern for IL-6 and TNF- and attributed this to a
localization of these proteins in the Golgi zone. After 24 hours of
stimulation, these proteins were distributed evenly within the cell,
probably due to the intracellular transport to the cell membrane. No
granulated staining was found for IL-1 (Fig 1D), again confirming
results by others who could not localize IL-1 in the endoplasmic
reticulum.53,54 Unlike most secreted proteins, IL-1 has
no signal sequence and is secreted via a pathway different from the
classical endoplasmic reticulum-Golgi route. Rubartelli et
al55 suggested that IL-1 is contained partly within
intracellular vesicles, which protect it from protease digestion. In
contrast to the proinflammatory cytokines, which were induced by LPS,
the staining for hematopoietic growth factors, G- and GM-CSF, was similar in unstimulated and LPS-stimulated cultures (shown for G-CSF in
Fig 1F and G and Table 1). Furthermore, levels of secreted G- and
GM-CSF appeared in the supernatant after stimulation with LPS. This may
be due to an induction of secretion by LPS (Table 2).
The analysis of cytokine protein and mRNA expression at the single cell
level in the MO/MAC cultures showed that even though the secreted
cytokine levels were several-fold higher in the supernatant, there were
no significant changes in the levels of mRNA and protein expression
within the infected cells. Others have shown that intracellular expression of IL-1 and TNF- in MO/MACs recovered from
HIV-1-infected and uninfected individuals were not significantly
different.56 Thus, an altered level of cytokine secretion
without a simultaneous change in the intracellular cytokine level
implies that other mechanisms such as the cytokine turnover in
individual cells and/or posttranslational processing must be
taking place. Posttranslational mechanisms have been reported for
TNF- and IL-1.57,58 For TNF-, it has been shown
that IL-1-stimulated production in the cytotrophoblastic cell line
BeWo is independent from TNF- mRNA induction and de novo protein
synthesis.57 IL-1 release from human monocytes is
promoted by adenosine triphosphate (ATP)-dependent intracellular ionic changes.58
By using double-labelling immunocytochemistry to stain intracellular
HIV-1 p24 and in situ hybridization to identify cytokine mRNA, we
showed that p24-positive MAC express similar amounts of proinflammatory
cytokine mRNA when compared with p24-negative MAC (Fig 2B, Table 3).
Our data indicate that HIV-1 infection most likely acts in an indirect
way leading to an altered cytokine production in the entire MAC
culture. The question is how HIV is altering the cytokine production
not only in the HIV-1-infected cells, but in the whole culture? One
possible explanation would be the paracrine action of HIV-1
transactivating protein Tat, which is released by infected cells and
taken up by uninfected cells.59,60 Tat can induce TNF-,
IL-1, IL-1, and interferon (IFN)- in a
dose-dependent manner.61,62 This effect is not only due to
transcriptional, but also translational control. Experiments by
Braddock et al63 showed that Tat increased the efficiency of translation. This was originally confirmed by the observation that
the increase in protein synthesis exceeds the increase of mRNA. An
enhanced secretion of proinflammatory cytokines may be due to an
accelerated transport from the inside of the cells to the outside. In
addition, it has been shown that Tat can bind to and inhibit the
dipeptidyl peptidase IV (CD26),64 an enzyme that is
expressed on monocytic cells.65 This enzyme catalyzes the
hydrolysis of cytokines with specific N-terminal peptide sequences, like TNF- or IL-6.66 Thus, Tat could increase the level
of cytokines in the supernatant by inhibiting the peptidase activity and thereby inhibiting cytokine degradation. For TNF-, it has been
shown that dipeptidyl peptidase IV activity regulates the extracellular
TNF- concentration.67,68
Here, we have shown that individually HIV-1-infected cells are not
directly responsible for the altered levels of secreted cytokine
levels, but may exert their action in an indirect manner. This could
explain how a low number HIV-1-infected cells (eg, in brain tissue)
contribute to HIV-1-related neurologic disorders or immune
dysfunction, respectively, by disturbing the cytokine balance.
| |
FOOTNOTES |
Submitted July 2, 1997;
accepted February 3, 1998.
Supported by a grant from the Bundesministerium für Bildung,
Wissenschaft, Forschung und Technologie (DLR: III-004-89/FVP4 and
01KI9411). The Georg-Speyer-Haus is supported by the Bundesministerium für Gesundheit and the Hessische Ministerium für
Wissenschaft und Kunst.
Address reprint requests to Reinhard Andreesen, MD,
Department of Hematology and Oncology, University of Regensburg,
Franz-Josef-Strauss-Allee 11, D-93055 Regensburg, Germany.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge Silke Deckert for excellent technical
assistance; C. Galanos, Freiburg, Germany, for LPS; and M. Ceska,
Vienna, Austria and G.R. Adolf, Vienna, Austria, for providing antibodies. For statistical analysis, we thank U. Alex.
| |
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Abstract
Introduction
Abstract