![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Virology, August 2000, p. 6849-6855, Vol. 74, No. 15
0022-538X/00/$04.00+0
Cathepsin G, a Neutrophil-Derived Serine Protease,
Increases Susceptibility of Macrophages to Acute Human Immunodeficiency
Virus Type 1 Infection
Hiroyuki
Moriuchi,1,2,3,*
Masako
Moriuchi,1,2 and
Anthony S.
Fauci1
Laboratory of Immunoregulation, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892,1 and Division
of Medical Virology, Department of Molecular Microbiology and
Immunology, Nagasaki University Graduate School of Medical Science,
Nagasaki 852-8123,2 and Department of
Pediatrics, Nagasaki University School of Medicine, 1-7-1 Sakamoto,
Nagasaki 852-8501,3 Japan
Received 23 December 1999/Accepted 4 May 2000
 |
ABSTRACT |
Neutrophils dominate acute inflammatory responses that generally
evolve into chronic inflammatory reactions mediated by
monocyte/macrophages and lymphocytes. The latter cell types also serve
as major targets for human immunodeficiency virus type 1 (HIV-1). In
this study we have investigated the role of neutrophil products,
particularly cathepsin G, in HIV infection. Cathepsin G induced
chemotaxis and production of proinflammatory cytokines by macrophages
but not CD4+ T cells. Pretreatment with cathepsin G
markedly increased susceptibility of macrophages but not
CD4+ T cells to acute HIV-1 infection. When macrophages
were exposed to pertussis toxin prior to cathepsin G treatment, the
cathepsin G-mediated effect was almost abrogated, suggesting that
enhancement of HIV-1 replication by cathepsin G requires Gi
protein-mediated signal transduction. Although prolonged exposure to
cathepsin G suppressed HIV infection of macrophages, serine protease
inhibitors, which are exuded from the bloodstream later during
inflammatory processes, neutralized the inhibitory effect. Neutrophil
extracts or supernatants from neutrophil cultures, which contain
cathepsin G, had effects similar to purified cathepsin G. Thus,
cathepsin G, and possibly other neutrophil-derived serine proteases,
may have multiple activities in HIV-1 infection of macrophages,
including chemoattraction of monocyte/macrophages (HIV-1 targets) to
inflamed tissue, activation of target cells, and increase in their
susceptibility to acute HIV-1 infection.
| |
INTRODUCTION |
Serine proteases constitute a gene
superfamily of proteolytic enzymes that are characterized by a unique
reactive serine side chain and that maintain critical and diverse
biological functions. In particular, it has recently been demonstrated
that proteases are capable of transducing outside-in signals of
leukocyte activation that influence a wide array of leukocyte effector
functions, such as cytotoxicity, chemotaxis, and costimulation of
cellular proliferation (reviewed in reference 1). A
specific family of lysosomal serine proteases (also called
serprocidins), including cathepsin G, elastase, and proteinase 3, are
expressed predominantly in neutrophils and, to a lesser extent, in
cells of the monocyte/macrophage lineage (6, 24, 29).
Neutrophils dominate acute inflammatory responses induced by microbial
infections or tissue damage. Subsequently, monocyte/macrophages and
lymphocytes mediate subacute and chronic inflammatory reactions. Recently, cathepsin G, a neutrophil-derived serine protease, was identified as a chemoattractant for monocytes (7). Cathepsin G was also shown to stimulate lymphocytes (12). Since
lymphocytes and monocytes are major targets of human immunodeficiency
virus type 1 (HIV-1), we sought to determine whether cathepsin
G-induced stimulation of these cells could modulate HIV infection.
In this study we demonstrate that (i) cathepsin G induces the
expression of a number of cytokines as well as chemotactic activity in
macrophages but not in CD4+ T cells; (ii) pretreatment of
macrophages with cathepsin G markedly increases susceptibility to HIV-1
infection, while cathepsin G has minimal effects on HIV-1 infection of
CD4+ T cells; (iii) prolonged exposure of macrophages to
cathepsin G suppressed HIV expression, but 
1-antichymotrypsin (ACT),
an inhibitor of cathepsin G present in the plasma, neutralizes the inhibitory effect mediated by prolonged exposure to cathepsin G; and
(iv) neutrophil extracts or supernatants from neutrophil cultures
containing cathepsin G have similar effects on HIV infection of
macrophages as does purified cathepsin G. These results suggest that
cathepsin G, and possibly other serine proteases, plays a critical role
in HIV infection of cells of the monocyte/macrophage lineage.
| |
MATERIALS AND METHODS |
Reagents.
Human neutrophil cathepsin G, elastase, and ACT
were purchased from ICN Pharmaceuticals Inc. (Costa Mesa, Calif.); the
purity of these reagents was >98% as determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (according to the
manufacturer's instructions). Human secretary leukocyte protease
inhibitor (SLPI) (purity, >97%) was purchased from R&D Systems
(Minneapolis, Minn.). Human macrophage-derived chemokine (MDC) was
purchased from Pepro Tech EC Ltd. (London, England). Pertussis toxin
(PTx) was purchased from Sigma Chemical (St. Louis, Mo.). Endotoxin was
undetected in these reagents by the Limulus amebocyte lysate
assay (BioWhittaker, Walkersville, Md.).
Cells.
Monocyte-derived macrophages (MDM) were isolated from
healthy volunteers (Department of Transfusion Medicine [DTM], Warren Grant Magnusson Clinical Center [WGMCC], National Institutes of Health [NIH], Bethesda, Md.) and propagated in Dulbecco's minimal essential medium (DMEM) supplemented with 10% human male AB serum (HS;
Sigma Chemical Co.), as described previously (17), and either unstimulated or stimulated with lipopolysaccharide (LPS) for 5 to 7 days before experiments. CD4+ T cells were prepared
from peripheral blood lymphocytes (PBL) as described previously
(19) and either unstimulated or stimulated with
phytohemagglutinin (PHA) for 3 days before experiments.
Granulocyte packs obtained from healthy donors were provided by the DTM
(WGMCC, NIH), and neutrophils were isolated as described previously
(5). The neutrophils were resuspended in AIM-V serum-free medium at 2 × 106 cells/ml and either unstimulated or
stimulated with LPS (100 ng/ml). After incubation at 37°C for 24 h, cell-free supernatants were collected and kept at 
80°C until
use. Neutrophil pellets were washed three times in phosphate-buffered
saline (PBS), resuspended in AIM-V medium at 2 × 106
cells/ml, subjected to three cycles of freezing-thawing, and centrifuged to remove cell debris, and crude cell extracts were kept at
80°C until use.
Viruses and infection.
T-cell-tropic (X4) NL4-3 and
macrophage-tropic (R5) ADA8 virus strains were propagated by
transfecting 293T cells with the respective molecular clone as
described previously (16), except that the cells were
maintained in serum-free AIM-V medium. Approximately 2 × 105 MDM were extensively washed in PBS, resuspended in
serum-free AIM-V medium (Life Technologies, Inc., Grand Island, N.Y.)
with or without cathepsin G, incubated for 30 min at 37°C, and then infected with the indicated molecular clone stock at a multiplicity of
infection of approximately 0.05. After a 3-h incubation at 37°C, the
infected cells were extensively washed in PBS, resuspended in AIM-V
with or without cathepsin G, and plated on a 96-well tissue culture
plate. Approximately half of the cell culture supernatants was
collected for the reverse transcriptase (RT) assay every 3 to 4 days.
Single-round virus replication assays.
Single-round virus
replication assays were performed using replication-incompetent
luciferase reporter viruses that were pseudotyped by Env derived from
either T-tropic (X4) HIV-1 HXB2, M-tropic (R5) HIV-1 ADA, or
amphotropic murine leukemia virus (AMV), as described previously
(10, 16). In brief, 5 × 105 cells were
extensively washed in PBS, resuspended in AIM-V with and without the
indicated serine protease, incubated for 30 min at 37°C, and then
infected with the indicated virus stock (50,000 cpm of RT activity).
Three days later, the infected cells were lysed and subjected to
luciferase assays, using commercially available reagents (Promega,
Madison, Wis.).
Transient-expression assays.
Freshly isolated monocytes were
plated on 60-mm-diameter tissue culture dishes and allowed to
differentiate into macrophages with DMEM supplemented with 10% HS for
5 to 7 days. The cells were transfected with 15 µg of pHIV-1 LTR-luc
(encoding the luciferase gene under the control of HIV-1 long terminal
repeat [LTR]) or 3 µg of pCMV-luc (encoding the luciferase gene
under the control of human cytomegalovirus [CMV] major
immediate-early gene promoter [MIEP]) by the modified calcium
phosphate method (20), washed, and incubated with AIM-V
medium in the presence of cathepsin G with and without ACT. Luciferase
activity was measured for cell lysates 2 days after transfection, as
described previously (18).
Flow cytometric analyses.
Fluorescein isothiocyanate (FITC)-
or phycoerythrin (PE)-conjugated antibodies to CCR5 and CXCR4 were
purchased from PharMingen (San Diego, Calif.), while FITC- and
PE-conjugated isotype controls were purchased from Becton-Dickinson
(San Jose, Calif.). Cells were stained with the indicated antibodies
and analyzed in a FACScan (Becton-Dickinson), as described previously
(16).
ELISA.
For the enzyme-linked immunosorbent assays (ELISA),
levels of tumor necrosis factor alpha (TNF-) and interleukin
(IL)-1
in the cell culture supernatants were measured using
commercially available ELISA kits (R&D Systems).
Chemotaxis assays.
Macrophage migration was evaluated using
a 96-well microchamber method, as described previously (16).
In brief, various amounts of either cathepsin G, elastase, or MDC were
added to 27.5 µl of RPMI 1640 supplemented with bovine serum albumin
(2 mg/ml) (RPMI-BSA) and placed in the lower wells of the chemotaxis chamber (Neuro Probe Inc., Cabin John, Md.), while cells were suspended
in 25 µl of RPMI-BSA at 2 × 106 cells/ml and placed
in the upper wells. After 2 h of incubation at 37°C, the
Nucleopore polycarbonate filter (5-µm pore size) that separated the
two compartments was removed, fixed, and stained, and the number of
cells migrating to the lower surface of the filter was counted under
the microscope. The mean number of migrating cells was calculated from
four high-power fields (HPF) (×400).
| |
RESULTS |
Cathepsin G is a chemoattractant for and inducer of cytokine
production by macrophages.
Certain members of the serine protease
family are capable of stimulating leukocytes to exert a wide array of
effector functions, such as cytotoxicity, chemotaxis, and costimulation
of cellular proliferation (reviewed in reference 1).
Therefore, we investigated the effects of cathepsin G, a
neutrophil-derived serine protease, on macrophages and lymphocytes, two
major targets for HIV-1.
First, chemotaxis assays showed that cathepsin G weakly chemoattracted
MDM (Fig. 1) and barely chemoattracted
monocytes (data not shown). The chemotactic activity of cathepsin G was
also reported by Chertov et al. (7). Furthermore, we have
demonstrated that cathepsin G attracted MDM stimulated with LPS, a
gram-negative bacterial cell wall component, much more efficiently than
unstimulated MDM (Fig. 1). These results suggest that the inflammatory
products of bacteria and neutrophils may synergize to attract MDM. In
contrast, cathepsin G did not efficiently chemoattract lymphocytes
whether unstimulated or stimulated with PHA (Fig. 1). Thus, the
chemotactic activity of these serine proteases appears to be specific
for MDM.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 1.
Cathepsin G is a chemoattractant for macrophages. MDM
were either unstimulated or stimulated with LPS for 3 days, while PBL
were either unstimulated or stimulated with PHA for 3 days. Cell
migration was evaluated using a 96-well microchamber technique as
described in Materials and Methods. The results are expressed as the
chemotaxis index, which represents the ratio of the number of migrating
cells in response to cathepsin G or MDC to those in control medium.
Baseline migration for unstimulated MDM, LPS-stimulated MDM,
unstimulated PBL, and PHA-stimulated PBL was 68 (29 to 89), 15 (5 to
29), 9 (2 to 18), and 6 (2 to 11) cells per HPF, respectively [mean
(range)]. Data represent means ± standard error of the mean
(SEM) for three independent experiments.
|
|
Next, we investigated whether stimulation of MDM with cathepsin G can
induce production of the proinflammatory cytokines TNF- and IL-1,
which have been shown to induce expression of HIV (reviewed in
reference 23). To this end, 106 MDM were
extensively washed in PBS, resuspended in AIM-V with or without
cathepsin G, incubated for 30 min at 37°C, washed in PBS, resuspended
in 1 ml of DMEM-10% HS, and incubated at 37°C. The supernatants
were collected 16 h after mock or cathepsin G treatment, and
levels of cytokines secreted from MDM were measured by ELISA. Exposure
to cathepsin G induced production of these cytokines by MDM but not by
CD4+ T cells (Table 1). Thus,
cathepsin G is a chemoattractant for and inducer of cytokine production
by macrophages.
Macrophages treated with cathepsin G become highly susceptible to
acute HIV infection.
Macrophages migrate to infected and inflamed
tissues and are exposed to microbial and neutrophil products, including
neutrophil-derived serine proteases such as cathepsin G. As indicated
above, cathepsin G can influence macrophage functions in various ways.
Thus, we sought to determine whether cathepsin G has an effect on HIV
infection of macrophages.
MDM were treated with cathepsin G and infected with HIV-1; HIV-1
replication was monitored by RT assays. As previously reported (21), replication of T-tropic (X4) HIV-1 was enhanced, while that of M-tropic (R5) HIV-1 was suppressed in LPS-stimulated MDM compared to unstimulated MDM (Fig. 2A).
Replication of both R5 and X4 HIV-1 was markedly enhanced in cathepsin
G-treated MDM compared to untreated MDM (Fig. 2A). In contrast,
cathepsin G had no effect on HIV infection of CD4+ T cells
(Fig. 2B). We also examined whether cathepsin G treatment of virus
stocks instead of target cells has any effect. 293T cells were
transfected with pNL4-3 or pAD8 and incubated with AIM-V serum-free
medium. The culture supernatants containing NL4-3 or ADA8 virus were
either mock treated or treated with cathepsin G for 30 min and then
mixed with RPMI 1640 supplemented with 20% FBS to neutralize the
enzymatic activity. Macrophages were infected with either mock-treated
or cathepsin G-treated virus stocks. The infectability of the virus was
not increased by cathepsin G treatment (data not shown). These results
indicate that cathepsin G interacts with a host factor(s), not virus
itself, to mediate its effects on HIV infection of macrophages.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 2.
Cathepsin G treatment renders macrophages (Mø) highly
susceptible to acute HIV-1 infection. MDM (A) and CD4+ T
cells (B) were either untreated or treated with cathepsin G (5 µg/ml)
for 30 min before infection with the indicated strain of HIV-1. Peak RT
titers on day 12 postinfection are shown. Results are representative of
seven independent experiments. (C) MDM either untreated or treated with
cathepsin G were infected with replication-incompetent luciferase
reporter NL4-3luc-R E virus that had been
pseudotyped by Env from either T-tropic HIV-1 HXB2, M-tropic HIV-1 ADA,
or AMV. Luciferase activity was measured for the infected cell lysates
3 days after infection. Results are representative of seven independent
experiments.
|
|
In order to further investigate the effects on HIV infection mediated
by cathepsin G, single-round virus replication assays were performed.
Similar to standard infection assays described above, pretreatment of
macrophages with cathepsin G enhanced the infectivity of all HIV-1
strains, including a strain pseudotyped by Env from AMV (Fig. 2C).
These results suggest that cathepsin G treatment of macrophages may
have multiple effects on HIV-1 infection during the viral replicative
cycle and that this effect appears to be independent of HIV
coreceptor-mediated entry, since infection even by amphotropic virus
was enhanced by treatment of macrophages with cathepsin G. Flow
cytometric analysis showed that cathepsin G treatment does not increase
expression of HIV coreceptors CCR5 and CXCR4 (Fig.
3).
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 3.
Cathepsin G (CatG) treatment does not increase cell
surface expression of HIV-1 coreceptors. MDM were either unstimulated
(control) or stimulated with cathepsin G (5 µg/ml) for 30 min, washed
twice, stained with anti-CXCR4 or anti-CCR5, and analyzed in a FACScan
as described in Materials and Methods. Filled areas indicate isotype
control staining, while open areas under solid lines indicate staining
with anti-CXCR4 or anti-CCR5. Results are representative of three
independent experiments.
|
|
Chemotactic activity or effect of cathepsin G on HIV-1 infection of
macrophages is dependent on its catalytic activity and Gi protein
signal transduction.
Some of the serine proteases are able to
transduce outside-in signals of leukocyte activation that influence a
wide array of leukocyte effector functions (reviewed in reference
1). A prototypic serine protease thrombin is capable
of transducing signals through the interaction with thrombin
receptors that are members of the
seven-transmembrane-domain, G-protein-coupled receptor family
(reviewed in reference 1). Proteolytic modification of thrombin receptors by thrombin appears to be critical for the events
mediated by thrombin (1). Although a receptor(s) for cathepsin G has not been well described, by analogy with thrombin, it
is possible that cathepsin G mediates its biological activities through
interaction with a seven-transmembrane-domain, G-protein-coupled receptor(s).
In order to address this hypothesis, the following experiments were
performed. First, cathepsin G-induced migration of macrophages was
determined following treatment of macrophages with PTx, an inhibitor of
Gi protein signal transduction (22), or pretreatment of
cathepsin G with ACT, a serine protease inhibitor present in plasma. As
shown in Fig. 4A, both PTx and ACT
markedly attenuated the chemotactic activity of cathepsin G.
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 4.
Chemotactic activity and effect of cathepsin G (CatG) on
HIV-1 infection of macrophages is dependent on its catalytic activity
and Gi protein signal transduction. (A) Chemotaxis assay. Where
indicated, macrophages were preincubated with PTx (300 ng/ml) for 15 min or cathepsin G was pretreated with ACT (10 µg/ml) prior to
treatment of macrophages. As controls, macrophages were treated with
ACT alone or PTx alone. Data represent mean ± SEM for three
independent experiments. (B) Infection assay. Where indicated,
macrophages were preincubated with PTx (300 ng/ml) for 15 min and
treated with cathepsin G (5 µg/ml) for 30 min. Cathepsin G was either
untreated or pretreated with ACT (10 µg/ml). Macrophages were then
infected with replication-incompetent luciferase reporter
NL4-3luc-RE virus that had been pseudotyped
by Env from M-tropic HIV-1 ADA. Luciferase activity was measured for
the infected cell lysates 3 days after infection. Data represent
mean ± SEM for five independent experiments.
|
|
Next, macrophages were exposed to ACT or PTx prior to cathepsin G
treatment, and levels of proinflammatory cytokines in the culture
supernatants were determined. As shown in Table 1, pretreatment of
macrophages with ACT or PTx abolished induction of proinflammatory cytokine (TNF- and IL-1) production.
Finally, macrophages were exposed to PTx prior to cathepsin G treatment
followed by infection with a replication-incompetent luciferase
reporter virus that had been pseudotyped by HIV-1 ADA Env. Pretreatment
of macrophages with cathepsin G markedly enhanced infectability by ADA
Env-bearing virus, and PTx treatment alone had no or minimal effect on
the infectivity; however, PTx treatment almost abrogated the cathepsin
G-mediated effect (Fig. 4B). Pretreatment of cathepsin G with ACT also
abrogated induction of viral replication by cathepsin G (Fig. 4B).
Taken together, these results suggest that cathepsin G mediates the
above biological functions through signal-transducing events following
its catalytic interaction with a seven-transmembrane-domain,
G-protein-coupled receptor(s).
Prolonged exposure to cathepsin G diminishes HIV-1 expression from
infected macrophages, but serine protease inhibitors in the plasma
neutralize the inhibitory effect.
The above experiments have
demonstrated that pretreatment of macrophages with cathepsin G enhanced
HIV-1 replication. In order to investigate the effect of prolonged
exposure to cathepsin G on HIV-1 infection, macrophages were treated
with cathepsin G at various time points and durations after infection
with HIV-1. In contrast to pretreatment with cathepsin G, HIV-1
infection of macrophages was suppressed by posttreatment with cathepsin G, and the inhibitory effect was dose and duration dependent (Fig. 5A
and B). Single-round virus replication
assays indicate that the inhibitory effect of cathepsin G is
independent of coreceptor-mediated cellular entry of the virus, since
infection even by amphotropic virus was suppressed by posttreatment of
macrophages with cathepsin G (Fig. 5C). Furthermore,
transient-expression assays indicate that cathepsin G treatment
downregulates HIV-1 LTR activity (Fig. 5D), while it had little effect
on transcription from CMV MIEP (data not shown).
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 5.
Prolonged exposure to cathepsin G downregulates HIV
expression. The inhibitory effect by postexposure to cathepsin G is
duration (A) and dose (B) dependent. (A) Macrophages were either mock
treated or treated with cathepsin G (5 µg/ml) immediately after or
for 30 min prior to infection with ADA8. Cathepsin G was washed out
after the indicated period (30 min or 24 h) of incubation or
continuously supplied by replacing approximately half of the medium
containing the same amount of cathepsin G every 4 days. Data represent
means ± SEM for three parallel experiments. Similar results were
obtained for three independent experiments. (B) Macrophages were
treated with the indicated amount of cathepsin G immediately after
infection with ADA8. Data represent means ± SEM for three
parallel experiments. Similar results were obtained for three
independent experiments. (C) The inhibitory effect of cathepsin G is
independent of coreceptor-mediated cellular entry of the virus.
Macrophages were either mock treated or treated with cathepsin G (5 µg/ml) for 24 h immediately after infection with
NL4-3luc-RE virus that had been
complemented with AMV Env. Data represent means ± SEM for five
independent experiments. (D) Postexposure to cathepsin G downregulates
HIV-1 LTR activity. Macrophages were either mock treated or treated
with cathepsin G for 24 h immediately after transfection with
pHIV-1 LTR-luc or pCMV-luc. Luciferase activity in the pHIV-1
LTR-luc-transfected cell lysates is shown. Data represent means ± SEM for five independent experiments.
|
|
As the inflammatory process proceeds, plasma is exuded into the
inflamed tissue, secondary to increased capillary permeability. A
number of serine protease inhibitors are present in the plasma and
other body fluids and play a critical role in the regulation of serine
protease activity (reviewed in reference 27). In
order to mimic the in vivo inflammatory process during which serine protease activity is likely to be neutralized by serine protease inhibitors in plasma, ACT, a serine protease inhibitor present in
plasma, was added at different time points to HIV-1-infected macrophage
cultures that had been treated with cathepsin G. Cathepsin G was added
30 min before and immediately after infection and replaced on days 4 and 8 postinfection. ACT was added either 30 min before and immediately
after infection [CatG+ACT (30 min)], immediately after infection
[CatG+ACT (+0 h)], or 24 h after infection [CatG+ACT (+24 h)]
and replaced on days 4 and 8 postinfection in either case. When
cathepsin G alone was added before and after infection in the absence
of ACT (CatG in Fig. 6), HIV replication was modestly higher on day 4 than in the control, probably reflecting the enhancing effect of cathepsin G; however, HIV replication was not
increased thereafter, indicating that cathepsin G posttreatment inhibited HIV replication. When ACT was added for the whole period with
cathepsin G [CatG+ACT (30 min) in Fig. 6], ACT neutralized both the
enhancing and inhibitory effects on HIV infection of macrophages by
cathepsin G, and there was no or minimal effect on infectability by
HIV-1 compared to the control. When ACT was added immediately after
virus absorption [CatG+ACT (+0 h) in Fig. 6], only the inhibitory
effect by cathepsin G was neutralized, and HIV replication was markedly
enhanced. When ACT was added 24 h after infection [CatG+ACT (+24
h) in Fig. 6], HIV replication was also enhanced, although to a lesser
degree, probably because the inhibitory effect of cathepsin G during
24 h after infection reduced its enhancing effect on HIV
replication. We have obtained similar results using other serine
protease inhibitors such as SLPI (data not shown). Therefore, it is
likely that the inflammatory process in vivo provides a favorable
environment for HIV infection: first, cathepsin G and other
inflammatory products (i.e., proinflammatory cytokines) accelerate
establishment of acute HIV infection, and subsequently serine protease
inhibitors neutralize the inhibitory effect by cathepsin G.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 6.
Serine protease inhibitor ACT neutralizes the effects
mediated by cathepsin G. Macrophages were treated with cathepsin G
(CatG, 5 µg/ml) for 30 min before and immediately after infection
with the ADA8 strain of HIV-1. ACT was added either 30 min before and
immediately after infection [CatG+ACT (30 min)], immediately after
infection [CatG+ACT (+0 h)], or 24 h after infection [CatG+ACT
(+24 h)]. Approximately half of the medium was replaced with AIM-V
medium containing the same amounts of cathepsin G and ACT every 4 days,
and RT activities in the cell-free supernatants were measured. Arrows
indicate time points when ACT was added. Solid and broken lines
indicate durations of cathepsin G treatment in the absence and presence
of ACT, respectively. Results are representative of four independent
experiments.
|
|
Neutrophil extracts or supernatants from neutrophil cultures had
similar effects on HIV infection of macrophages as did cathepsin
G.
We next investigated whether crude extracts from neutrophils or
supernatants from neutrophil cultures which contain cathepsin G and
other serine proteases similarly modulate HIV infection. Neutrophils
were either unstimulated or stimulated with LPS, which is known to
induce the production and release of cathepsin G. Pretreatment of
macrophages with these neutrophil-derived products enhanced HIV-1
replication (Fig. 7), while posttreatment
suppressed HIV-1 infection of macrophages (data not shown). In the
supernatant transfer experiments, LPS added to neutrophil cultures
might be transferred to macrophage cultures and could influence HIV-1
ADA8 infection of macrophages (see Fig. 2), neutrophil-derived factors appeared to overcome possibly inhibitory effects of LPS. Serine protease inhibitors neutralized both enhancing and inhibitory effects
by neutrophils (Fig. 7; data not shown). These results indicate that
neutrophils can modulate HIV infection by producing serine proteases,
which include but may not be limited to cathepsin G.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 7.
Neutrophil extracts (Neu Ext) or crude supernatants from
neutrophil cultures (Neu Sup) render macrophages highly susceptible to
acute HIV-1 infection. Macrophages were either mock treated (Unstim.)
or treated with the indicated neutrophil derivatives for 30 min and
then infected with ADA8 virus. The infected cells were washed and
resuspended in AIM-V serum-free medium, and approximately half of the
culture supernatant was collected every 3 to 4 days for RT assays. Peak
titers on day 12 postinfection are shown. Results are representative of
four independent experiments.
|
|
| |
DISCUSSION |
Bacterial infections have been associated with increased HIV
transmission and replication (reviewed in references 4,
8, and 28). Since HIV infection is
associated with immune activation (reviewed in reference
11), stimulation of lymphocytes and macrophages, which serve as major target cells for HIV, has been considered to be
responsible for disease progression as well as efficient transmission
(reviewed in references 4, 8, and
28). In this regard, we have recently demonstrated
that exposure to bacterial cell wall components renders macrophages
highly susceptible to T-tropic or dual-tropic HIV-1 infection
(21), which predominates during the later stages of HIV
disease in certain individuals (9, 25).
Neutrophils are recruited to the site of most infections and dominate
acute inflammatory responses. Therefore, although neutrophils themselves do not serve as host cells for HIV, we hypothesize that
neutrophils or their products may play a role in the pathogenesis of
HIV disease, especially in patients with microbial coinfections. In
this regard, a recent study by Ho et al. (13) has
demonstrated that neutrophils are capable of increasing HIV replication
by the generation of reactive oxygen intermediates or by
proinflammatory cytokines; however, other neutrophil products may also
influence HIV infection. Among such neutrophil products is cathepsin G, which has been shown to chemoattract macrophages (7), to
stimulate lymphocytes (12), and to bind to the V3 loop of
HIV-1 gp120 (3). Therefore, we examined the role of
cathepsin G in HIV infection of macrophages and lymphocytes, which
dominate subacute to chronic inflammatory responses. We have confirmed
that cathepsin G is an efficient chemoattractant for macrophages and
have also shown that macrophages stimulated with LPS, a bacterial
product, migrate much more efficiently in response to cathepsin G than do unstimulated macrophages. We have also demonstrated that cathepsin G
induces expression of proinflammatory cytokines by macrophages and,
more importantly, increases the susceptibility of these cells to acute
HIV infection. Although prolonged exposure to cathepsin G suppressed
HIV expression, introduction of serine protease inhibitors counteracted
the inhibitory effect, a scenario that likely occurs during the
inflammatory process in vivo. These results indicate that cathepsin G,
a neutrophil product, may play a critical role in HIV infection of
macrophages in inflamed tissue: it chemoattracts macrophages (HIV
targets), activates them, and renders them highly susceptible to acute
HIV infection. We have obtained similar but less pronounced results for
another neutrophil-derived serine protease, elastase (data not shown).
Taken together, these observations suggest that some neutrophil-derived
serine proteases may play a critical role in HIV-1 infection of
macrophages in infected or inflamed tissue.
Although we have shown that cathepsin G-mediated signal-transducing
events appear to influence a postfusion/entry early event(s) during the
HIV replicative cycle, the precise mechanisms whereby cathepsin G
modulates HIV infection of macrophages remain unknown. While previous
studies have indicated that cathepsin G can interact with the V3 loop
of HIV-1 gp120, the present study suggests that interaction of
cathepsin G with a host factor(s) but not with virus itself is critical
for the effects mediated by the enzyme. Interestingly, SLPI, a natural
inhibitor of serine proteases including cathepsin G (26),
has been shown to suppress HIV infection of monocytes in vitro
(14). Although its mechanism of action remains to be
determined, SLPI appears to inhibit HIV replication at an early step(s)
during the viral replicative cycle by binding specifically to a host
cell molecule(s) other than CD4 (14, 15). Therefore, it is
possible that SLPI suppresses HIV infection of monocytes by
counteracting a serine protease(s) which is capable of enhancing HIV
replication in these cells. Increased infectability of murine retrovirus by protease treatment of host cells has also been reported (2). Taken together, these studies suggest that proteases
and protease inhibitors may play an important role in the pathogenesis of retroviral diseases. Identification of the host factor(s)
responsible for the protease-mediated effects is currently under investigation.
| |
ACKNOWLEDGMENTS |
We thank M. Martin (NL4-3), T. Theodore (AD8), and N. Landau
(NL4-3luc-RE virus) for reagents (in
parentheses) and J. Weddle for graphic work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatrics, Nagasaki University School of Medicine, 1-7-1 Sakamoto,
Nagasaki 852-8501, Japan. Phone: 81-95-849-7297. Fax: 81-95-849-7301. E-mail: hiromori{at}net.nagasaki-u.ac.jp.
| |
REFERENCES |
| 1.
|
Altieri, D. C.
1995.
Proteases and protease receptors in modulation of leukocyte effector functions.
J. Leukocyte Biol.
58:120-127[Abstract].
|
| 2.
|
Anderson, K. B., and H. Skov.
1989.
Retrovirus-induced cell fusion is enhanced by protease treatment.
J. Gen. Virol.
70:1921-1927[Abstract/Free Full Text].
|
| 3.
|
Avril, L. E.,
M. Di Martino-Ferrer,
G. Pignede,
M. Seman, and F. Gauthier.
1994.
Identification of the U-937 membrane-associated proteinase interacting with the V3 loop of HIV-1 gp120 as cathepsin G.
FEBS Lett.
345:81-86[CrossRef][Medline].
|
| 4.
|
Blanchard, A.,
L. Montagnier, and M.-L. Gougeon.
1997.
Influence of microbial infections on the progression of HIV disease.
Trends Microbiol.
5:326-331[CrossRef][Medline].
|
| 5.
|
Boyum, A.
1968.
Separation of leukocytes from blood and bone marrow.
Scand. J. Clin. Lab. Investig.
21(Suppl.):77-89[Medline].
|
| 6.
|
Campbell, E. J.,
E. K. Silverman, and M. A. Campbell.
1989.
Elastase and cathepsin G of human monocytes: quantification of cellular content, release in response to stimuli, and heterogeneity in elastase-mediated proteolytic activity.
J. Immunol.
143:2961-2968[Abstract].
|
| 7.
|
Chertov, O.,
H. Ueda,
L. L. Xu,
K. Tani,
W. J. Murphy,
J. M. Wang,
O. M. Howard,
T. J. Sayers, and J. J. Oppenheim.
1997.
Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils.
J. Exp. Med.
186:739-747[Abstract/Free Full Text].
|
| 8.
|
Cohen, M. S.
1998.
Sexually transmitted diseases enhance HIV transmission: no longer a hypothesis.
Lancet
351:sIII5-sIII7.
|
| 9.
|
Connor, R. L.,
K. E. Sheridan,
C. Ceradini,
S. Choe, and N. R. Landau.
1997.
Change in coreceptor use correlates with disease progression in HIV-1 infected individuals.
J. Exp. Med.
185:621-628[Abstract/Free Full Text].
|
| 10.
|
Deng, H.,
R. Liu,
W. Ellmeier,
S. Choe,
D. Unutmaz,
M. Burkhart,
P. Di Marzio,
S. Marmon,
R. E. Sutton,
C. M. Hill,
C. B. Davis,
S. C. Peiper,
T. J. Schall,
D. R. Littman, and N. R. Landau.
1996.
Identification of a major co-receptor for primary isolates of HIV-1.
Nature
381:661-666[CrossRef][Medline].
|
| 11.
|
Fauci, A. S.
1996.
Host factors and the pathogenesis of HIV-induced disease.
Nature
384:529-534[CrossRef][Medline].
|
| 12.
|
Hase-Yamaguchi, T., and Y. Aoki.
1995.
Stimulation of human lymphocytes by cathepsin G.
Cell. Immunol.
160:24-35[CrossRef][Medline].
|
| 13.
|
Ho, J. L.,
S. He,
A. Hu,
J. Geng,
F. G. Basile,
M. G. Almeida,
A. Y. Saito,
J. Laurence, and W. D. Johnson, Jr.
1995.
Neutrophils from human immunodeficiency virus (HIV)-seronegative donors induce HIV replication from HIV-infected patients' mononuclear cells and cell lines: an in vitro model of HIV transmission facilitated by Chlamydia trachomatis.
J. Exp. Med.
181:1493-1505[Abstract/Free Full Text].
|
| 14.
|
McNeely, T. B.,
M. Dealy,
D. J. Dripps,
J. M. Orenstein,
S. P. Eisenberg, and S. M. Wahl.
1995.
Secretary leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro.
J. Clin. Investig.
96:456-464.
|
| 15.
|
McNeely, T. B.,
D. C. Shugars,
M. Rosendahl,
C. Tucker,
S. P. Eisenberg, and S. M. Wahl.
1997.
Inhibition of human immunodeficiency virus type 1 infectivity by secretary leukocyte protease inhibitor occurs prior to viral reverse transcription.
Blood
90:1141-1149[Abstract/Free Full Text].
|
| 16.
|
Moriuchi, H.,
M. Moriuchi,
J. Arthos,
J. Hoxie, and A. S. Fauci.
1997.
Promonocytic U937 subclones expressing CD4 and CXCR4 are resistant to infection with and cell-to-cell fusion by T-cell-tropic human immunodeficiency virus type 1.
J. Virol.
71:9664-9671[Abstract].
|
| 17.
|
Moriuchi, H.,
M. Moriuchi,
C. Combadiere,
P. M. Murphy, and A. S. Fauci.
1996.
CD8+ T-cell-derived factor(s), but not -chemokines RANTES, MIP-1, and MIP-1, suppress HIV-1 replication in monocyte/macrophages.
Proc. Natl. Acad. Sci. USA
93:15341-15345[Abstract/Free Full Text].
|
| 18.
|
Moriuchi, H.,
M. Moriuchi, and A. S. Fauci.
1997.
Nuclear factor- B potently up-regulates the promoter activity of RANTES, a chemokine that blocks HIV infection.
J. Immunol.
158:3483-3491[Abstract].
|
| 19.
|
Moriuchi, H.,
M. Moriuchi, and A. S. Fauci.
1998.
Factors secreted by human T lymphotropic virus type I (HTLV-I)-infected cells can enhance or inhibit replication of HIV-1 in HTLV-I-uninfected cells: implications for in vivo coinfection with HTLV-I and HIV-1.
J. Exp. Med.
187:1689-1697[Abstract/Free Full Text].
|
| 20.
|
Moriuchi, M.,
H. Moriuchi,
S. E. Straus, and J. I. Cohen.
1994.
Varicella-zoster virus (VZV) virion-associated transactivator open reading frame 62 protein enhances the infectivity of VZV DNA.
Virology
200:297-300[CrossRef][Medline].
|
| 21.
|
Moriuchi, M.,
H. Moriuchi,
W. Turner, and A. S. Fauci.
1998.
Exposure to bacterial products renders macrophages highly susceptible to T-tropic human immunodeficiency virus type 1: implications for in vivo coinfections.
J. Clin. Investig.
109:1540-1550.
|
| 22.
|
Murphy, P. M.
1996.
Chemokine receptors: structure, function and role in microbial pathogenesis.
Cytokine Growth Factor Res.
7:47-64[CrossRef][Medline].
|
| 23.
|
Poli, G., and A. S. Fauci.
1995.
A role of cytokine in the pathogenesis of human immunodeficiency virus infection, p. 421-449.
In
B. Aggarwahl, and R. Puri (ed.), Human cytokines: their role in disease and therapy. Blackwell Science, Cambridge, Mass.
|
| 24.
|
Takahashi, H.,
T. Nukiwa,
P. Basset, and R. G. Crystal.
1988.
Myelomonocytic cell lineage expression of the neutrophil elastase gene.
J. Biol. Chem.
263:543-547.
|
| 25.
|
Tersmette, M.,
R. E. de Goede,
B. J. Al,
I. N. Winkel,
R. A. Gruters,
H. T. Cuypers,
H. G. Huisman, and F. Miedema.
1988.
Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytium-inducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex.
J. Virol.
62:2026-2032[Abstract/Free Full Text].
|
| 26.
|
Thompson, R. C., and K. Ohlsson.
1986.
Isolation, properties, and complete amino acid sequence of human secretary leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase.
Proc. Natl. Acad. Sci. USA
83:6692-6696[Abstract/Free Full Text].
|
| 27.
|
Travis, J., and G. S. Salvesen.
1983.
Human plasma protease inhibitors.
Annu. Rev. Biochem.
52:655-709[CrossRef][Medline].
|
| 28.
|
Wahl, S. M., and J. M. Orenstein.
1997.
Immune stimulation and HIV-1 viral replication.
J. Leukocyte Biol.
62:67-71[Abstract].
|
| 29.
|
Zimmer, M.,
R. L. Medcalf,
T. M. Fink,
C. Mattmann,
P. Lichter, and D. E. Jenne.
1992.
Three human elastase-like genes coordinately expressed in the myelomonocyte lineage are organized as a single genetic locus on 19pter.
Proc. Natl. Acad. Sci. USA
89:2228-2232.
|
Journal of Virology, August 2000, p. 6849-6855, Vol. 74, No. 15
0022-538X/00/$04.00+0
This article has been cited by other articles:
-
Geraghty, P., Greene, C. M., O'Mahony, M., O'Neill, S. J., Taggart, C. C., McElvaney, N. G.
(2007). Secretory Leucocyte Protease Inhibitor Inhibits Interferon-{gamma}-induced Cathepsin S Expression. J. Biol. Chem.
282: 33389-33395
[Abstract]
[Full Text]
-
Venkataraman, N., Cole, A. L., Svoboda, P., Pohl, J., Cole, A. M.
(2005). Cationic Polypeptides Are Required for Anti-HIV-1 Activity of Human Vaginal Fluid. J. Immunol.
175: 7560-7567
[Abstract]
[Full Text]
-
Berahovich, R. D., Miao, Z., Wang, Y., Premack, B., Howard, M. C., Schall, T. J.
(2005). Proteolytic Activation of Alternative CCR1 Ligands in Inflammation. J. Immunol.
174: 7341-7351
[Abstract]
[Full Text]
-
Gabali, A. M., Anzinger, J. J., Spear, G. T., Thomas, L. L.
(2004). Activation by Inflammatory Stimuli Increases Neutrophil Binding of Human Immunodeficiency Virus Type 1 and Subsequent Infection of Lymphocytes. J. Virol.
78: 10833-10836
[Abstract]
[Full Text]
-
Sun, R., Iribarren, P., Zhang, N., Zhou, Y., Gong, W., Cho, E. H., Lockett, S., Chertov, O., Bednar, F., Rogers, T. J., Oppenheim, J. J., Wang, J. M.
(2004). Identification of Neutrophil Granule Protein Cathepsin G as a Novel Chemotactic Agonist for the G Protein-Coupled Formyl Peptide Receptor. J. Immunol.
173: 428-436
[Abstract]
[Full Text]
Top
Abstract
Introduction
