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Department of Biology, Microbiology and Immunology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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s) mediate immunosuppression, in
part, through increased production of factors that suppress T cell
responsiveness and underproduction of positive regulatory cytokines.
Pretreatment of tumor-bearing host (TBH) Ms with the anticancer
agent paclitaxel (Taxol) partially reverses tumor-induced M
suppressor activity, suggesting that paclitaxel may restore TBH M
production of proimmune factors. Because paclitaxel demonstrates
LPS-mimetic capabilities and increased production of the LPS-induced
immunostimulatory cytokine IL-12 could account for enhanced T cell
responsiveness, we investigated whether paclitaxel induces M IL-12
production. Tumor growth significantly down-regulated M IL-12 p70
production through selective dysregulation of IL-12 p40 expression. LPS
stimulation failed to overcome tumor-induced dysregulation of p40
expression. In contrast, paclitaxel significantly enhanced both normal
host and TBH M IL-12 p70 production in vitro, although TBH M
IL-12 production was lower than that of similarly treated normal host
Ms. Paclitaxel enhanced p40 expression in a dose-dependent manner.
Through reconstituted M IL-12 expression, paclitaxel pretreatment
relieved tumor-induced M suppression of T cell alloreactivity.
Blocking M NO suppressed paclitaxel’s ability to induce IL-12
production. This suggests that paclitaxel-induced activities may
involve a NO-mediated autocrine induction pathway. Collectively, these
data demonstrate that paclitaxel restores IL-12 production in the TBH
and ascribe a novel immunotherapeutic component to the pleiotropic
activities of NO. Through its capacity to induce IL-12 production,
paclitaxel may contribute to the correction of tumor-induced immune
dysfunction. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)4 serve vital roles in
host defense against tumors, including tumor cytotoxicity and
stimulation of antitumor lymphocytes. However, tumors circumvent these
host-mediated immune activities through the production and release of
immunomodulatory factors, such as IL-10, PGE2, and
TGF-ß1, that adversely alter M function and phenotype. These
tumor-derived factors generate immunoregulatory Ms that inhibit T
cell responses and that are tumoricidally dysfunctional (reviewed in
1). Identification of the molecular and cellular origins of
tumor-induced changes in M function will increase opportunities for
immunotherapeutic intervention. Recent studies (2, 3, 4) ascribed
immune-activating properties to paclitaxel (Taxol), suggesting that
chemotherapeutic applications of paclitaxel may impart novel
immunotherapeutic activities on tumor-induced Ms.
A plant-derived diterpenoid (5), paclitaxel first gained recognition as
a potent inhibitor of cell cycle progression, leading to neoplastic
cell death and arrest of tumor progression (6, 7, 8). Paclitaxel
demonstrated significant antitumor efficacy in human clinical trials
(9) and became the chemotherapeutic agent of choice for first-line
treatment of metastatic breast cancers (10). Paclitaxel’s primary
mechanism of antineoplastic activity rests in its ability to
irreversibly polymerize 
/ß tubulin, thereby disrupting cellular
microtubule networks (11). Additionally, paclitaxel enhances tumor cell
radioresponse (12) and sensitivity to immune effectors such as TNF-
(13).
In addition to its well-characterized chemotherapeutic activities,
paclitaxel has profound cell cycle-independent effects on murine Ms
(2, 3, 4, 14). Paclitaxel induces normal host (NH) M responses similar
to those generated by bacterial LPS (15, 16), including enhanced NO (2, 17), TNF- (18), IL-1ß (19), and superoxide anion (20) production
and induction of NF-
B expression (21). Through increased TNF- and
NO production, paclitaxel enhances in vitro tumor cell cytotoxicity
(17).
Paclitaxel’s most significant immune activity, from a therapeutic
standpoint, may be its capacity to reverse tumor-induced M-mediated
immune suppressor activities. Paclitaxel pretreatment of tumor-bearing
host (TBH)-derived Ms modulated suppression of alloantigen-activated
T cell responsiveness (3) and concurrently enhanced M antitumor
cytotoxicity in vitro (3). Paradoxically, both the cytotoxic (3, 17)
and immunosuppressive (22) functions of TBH Ms are mediated
partially through the overproduction of reactive nitrogen and oxygen
intermediates, including NO, and proinflammatory cytokines, such as
TNF-. However, ablation of M-derived suppressor molecules NO and
TNF- failed to fully overcome the suppressive effects induced by
tumor growth in our model system (D. W. Mullins and K. D.
Elgert, unpublished observation), leading to speculation that TBH Ms
may be deficient in immunostimulatory factor production. The capacity
of the M-derived cytokine IL-12 to promote cell-mediated antitumor
immune responses during tumor growth (23, 24) and the LPS-mimetic
activities of paclitaxel (15, 16, 25, 26, 27, 28) suggested to us that
paclitaxel may reverse tumor-induced suppressor activities through
reconstitution of M IL-12 production in the TBH. We report that
fibrosarcoma growth dysregulates M production of IL-12, compromising
antitumor activity, and that paclitaxel induces IL-12 expression
through a NO-dependent autocrine induction mechanism.
Paclitaxel-activated Ms coexpress cytotoxic and immunostimulatory
activities that collectively may impart significant antitumor
immunotherapeutic functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 12-wk-old BALB/c (H-2d) male mice (Harlan
Sprague Dawley, Madison, WI) were used as source for normal host and
TBH lymphocytes. A BALB/c nonmetastatic methylcholanthrene-induced
transplantable fibrosarcoma, designated Meth-KDE, was used as described
(29). The use of a nonmetastatic tumor facilitates the study of
tumor-induced distal M populations. The Meth-KDE fibrosarcoma
induces significant systemic immunosuppression through the production
of the soluble suppressor cytokines IL-10, TGF-ß1, and
PGE2 (30). Tumors were induced by i.m. injection of 4
x 105 transplanted Meth-KDE cells, and palpable tumors
developed within 10 days. TBH BALB/c mice were used 21 days after tumor
passage because tumor-induced M suppressor activity is maximal at
this time (31), without cachexia or necrosis. Eight- to 12-wk-old male
C3H-HeJ (H-2k) mice were the source of alloantigen
stimulator cells. All procedures conformed to established National
Institutes of Health guidelines and were approved by the Virginia Tech
Animal Care Committee.
Medium and reagents
Lymphocytes were cultured in serum-free RPMI 1640 medium with 2 mM L-glutamine (Sigma, St. Louis, MO). All media contained 50 mg/L gentamicin sulfate (Tri-Bio Laboratories, State College, PA), 25 mM sodium bicarbonate (NaHCO3), and 25 mM HEPES buffer (Sigma). T cell cultures were supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA). RPMI 1640 medium was endotoxin free (<10 pg/ml) as assessed by the Limulus amebocyte lysate assay (Sigma).
Paclitaxel was dissolved in 100% DMSO (Mallinckrodt Chemical, Paris,
KY) to a 4 mM stock solution, stored at -80°C, and diluted to assay
concentrations in RPMI 1640 medium immediately before use. The final
concentration of DMSO in cultures was <1%. Bacterial LPS
(Escherichia coli serotype 026:B6) and the specific NO
inhibitor NG-monomethyl-L-arginine (NMMA;
Sigma) were reconstituted in RPMI 1640 and diluted to assay
concentrations immediately before use. Other reagents included
recombinant murine IFN-
(2.98 x 105 U/ml,
endotoxin content <0.1 ng/ml; Genzyme, Cambridge, MA), recombinant
murine IL-12 (sp. act. 2.5 x 106 U/ml, endotoxin
content <10 pg/ml; Genetics Institute, Cambridge, MA), and paclitaxel
(Calbiochem, La Jolla, CA). Neutralizing anti-IL-12 mAb (32) was
purified by ammonium sulfate fractionation (33) of spent medium from
C17.8 hybridoma cells (G. Trinchieri, Wistar Institute, Philadelphia,
PA) grown for 4 days in RPMI 1640 supplemented with 10% FBS. The mAb
fraction was concentrated by dialysis against fresh medium.
Neutralizing anti-IL-2 mAb (S4B6) and rat IgG2a isotype control mAb
were acquired from PharMingen (La Jolla, CA).
Immune cell collection and culture
Normal host and TBH peritoneal exudate Ms were collected by
lavage with cold RPMI 1640 medium, 4 days after i.p. injection with 2
ml of sterile 3% thioglycollate (Difco). Ms were purified by
plating peritoneal exudate cells for 2 h (37°C, 5%
CO2) in glass plates, washing away nonadherent cells with
warm RPMI 1640 medium, and collecting adherent Ms in cold medium by
scraping with a rubber policeman. Splenic Ms were collected by
plating pooled whole spleen cells for 2 h, washing away
nonadherent cells with warm medium, and collecting adherent Ms. The
final M preparations contained cells that were >95% viable and
>96% esterase positive, and flow cytometric analysis with M1/70 and
F4/80 mAbs (American Type Culture Collection (ATCC), Manassas, VA)
showed them to be >80% Mac-1+ and F4/80+,
respectively.
T cells were collected by plating normal host-derived whole spleen cells for 2 h in glass plates and collecting the nonadherent cell fraction. Purified CD4+ T cells were obtained by treatment with anti-CD8 (ATCC clone 3.155), anti-IAd (ATCC clone MK-D6), and anti-B cell and immature T cell (ATCC clone J11d) Abs and complement (Low-Tox rabbit complement; Accurate Chemical). RBC were lysed with 0.83% ammonium chloride (Sigma). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
IL-12 protein measurement
Either NH or TBH Ms (4 x 105 cells/well)
were cultured with the indicated reagents in 96-well flat-bottom
plates. Supernatants were collected and immediately assayed for IL-12
using a p70-specific ELISA (IL-12 DuoSet; R&D Systems, Minneapolis, MN)
per the manufacturer’s protocol. The ELISA consisted of anti-mouse
IL-12 p70 capture Ab adhered to high affinity protein binding plates
(Nunc (Naperville, IL) MaxiSorp ELISA plates), biotinylated secondary
Ab, and HRP-conjugated streptavidin detection reagent. ELISAs were
developed with Sigma tetramethylbenzidine liquid substrate system, and
absorbance was determined at 450 nm using an MR-600 microplate reader
(Dynex, Alexandria, VA). The limit of detection in our hands was
approximately 10 pg/ml.
Ribonuclease protection assay analysis of IL-12 subunit expression
Either NH or TBH Ms (5.0 x 106 cells) were
cultured in serum-free medium with the indicated reagents. Ms were
cultured for 2 h in 24-well flat-bottom plates (Corning Cell
Wells, Corning, NY), collected, and stored in liquid nitrogen for
subsequent analysis. Total mRNA was extracted using the RNeasy total
RNA system following cell disruption by passage through a QIAshredder
(both from Qiagen, Chatsworth, CA), per the manufacturer’s protocol.
Antisense riboprobes were constructed from specific IL-12 cDNA templates (PharMingen) using the MAXIscript T-7 in vitro transcription system (Ambion, Austin, TX) and labeled using the BrightStar psoralin-biotin method (Ambion). Ribonuclease protection assay (RPA) was performed using the Ambion RPA-II system, per the manufacturer’s protocol. RPA products were separated on a 5% polyacrylamide gel containing 8 M urea, transferred to nylon membrane, and visualized with the Ambion BioDetect nonisotopic detection system on Kodak (Rochester, NY) BioMax MR-1 autoradiography film. Images were digitized, and densitometric analyses were performed using NIH-Image software for Macintosh.
T cell alloreactivity assays
Either NH or TBH Ms (2 x 105 cells) were
added to NH T cell cultures (2 x 105 cells) in
96-well U-bottom tissue culture plates (Nunc). Some Ms were
pretreated with paclitaxel or LPS for 4 h before addition to T
cell cultures. Irradiated (2000 rad) whole splenic cell preparations
(4 x 105 cells) from C3H (H-2k) mice were
used as allogeneic stimulator cells. Neutralizing anti-IL-12 mAb
(C17.8, 10 µg/ml) or rat IgG2a isotype control mAb (10 µg/ml) was
added to some cultures. Eighteen hours before harvest, cultures were
pulsed with 1 µCi per well [3H]TdR, (sp. act. 6.7
Ci/mM; DuPont-NEN Research Products, Boston, MA). Cells were harvested
with a Skatron (Chantilly, VA) automated cell harvester and sample
activities determined using a Beckman Coulter (Fullerton, CA) LS 6000SC
scintillation counter.
Macrophage nitrite production
Following incubation, supernatants (100 µl) were collected
from alloantigen-activated T cell proliferation cultures. NO was
analyzed by measuring total nitrite levels in culture supernatants
using the Griess reagent, as described elsewhere (34). Briefly, 100
µl of M supernatants was added to 100 µl Griess reagent (0.1%
naphthylenediamine dihydrochloride, 1.0% sulfanilamide, 2.5%
H3PO4; Sigma) and incubated at room temperature
for 10 min; absorbance was read at 570 nm. A sodium nitrite (Sigma)
standard curve was used to calculate nitrite content in supernatants.
Nitrite was not detected in RPMI 1640 medium alone.
Statistics and calculations
Cells from 6 to 10 NH or TBH mice were pooled for each experiment. Triplicate cultures were tested in specific ELISAs, Griess reagent tests, and proliferation assays. Data are presented as means ± SEM of triplicate independent determinations. All experiments were repeated at least three times; representative experiments are shown. All comparisons were tested for significance by Student’s t test, and all comparisons are significant at the p < 0.05 level, unless otherwise stated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production of IL-12 p70 heterodimer
In the presence of tumor-induced Ms, T cell proliferative
responses are suppressed, suggesting that neoplastic tissues subvert
M function to favor tumor growth (1). In our model system,
tumor-derived factors such as PGE2, TGF-ß1, and IL-10
induce systemic dysregulation of M functions (30). Therefore, we
determined whether tumor growth compromises IL-12 production by Ms
derived from tumor-distal compartments. NH or TBH peritoneal Ms
(4 x 105 cells) were IFN- primed (10 U/ml) and
cultured without or with LPS (0.1–10.0 µg/ml) for 24 h.
Supernatants were collected and assayed for IL-12 heterodimer by
p70-specific ELISA (Fig. 1
). IL-12
production in the absence of IFN- priming was minimal (not shown),
consistent with the findings that the IL-12 p40 gene promoter is primed
by IFN- (35). Tumor growth significantly (p
< 0.05) inhibited IFN--induced IL-12 production (74% decrease
compared with NH Ms). LPS triggering stimulated NH M IL-12
production, leading to greater than a 120% increase in IL-12 at the
optimal dose of LPS (1.0 µg/ml). LPS enhanced IFN--primed TBH M
production of IL-12 by 115% at the optimal dose (1.0 µg/ml), as
compared with untriggered TBH Ms; however, the level of IL-12
production by TBH Ms was significantly (p <
0.005) less than similarly treated NH Ms at all doses of LPS tested.
TBH M IL-12 production, regardless of LPS triggering, never exceeded
that of untriggered NH Ms, demonstrating a serious lesion in the
response of TBH M populations. Splenic M cultures produced
similar profiles (not shown).
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IL-12 p40 expression
The bioactive IL-12 heterodimer is composed of the constitutively
expressed p35 subunit and the inducible p40 subunit (36). Because the
p70 heterodimer is necessary for biologic activity and p70 production
is regulated by the level of available p40, we determined whether tumor
growth dysregulates M p40 expression in our murine fibrosarcoma
model. Either NH or TBH peritoneal Ms (5 x 106
cells) were IFN- primed (10 U/ml) and cultured for 4 h (optimal
time) without or with LPS (1.0 µg/ml, optimal dose). Total RNA was
collected and p40 expression was measured by RPA (Fig. 2A). LPS failed to enhance p40
expression in tumor-induced Ms, further defining the lesion in TBH
M function. LPS did induce p40 expression in NH cells, consistent
with the reports of others (37), suggesting that tumor growth
dysregulates IL-12 heterodimer production through abrogated activation
response of the inducible p40 subunit. Expression of the housekeeping
gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was consistent
throughout all samples (Fig. 2B).
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IL-12 production
Paclitaxel has M-activating functions in vitro (2, 17) and in
vivo (3), and paclitaxel pretreatment of TBH Ms reverses
tumor-induced M suppression of T cell proliferation (3). Because
administration of exogenous IL-12 can promote antitumor immune function
and paclitaxel can activate expression of several LPS-inducible genes,
we hypothesized that reconstituted M IL-12 production may account
for restored T cell proliferative responses. Therefore, we determined
whether paclitaxel could induce M IL-12 production. NH or TBH
(4 x 105) peritoneal Ms were primed with IFN-
(10 U/ml) and cultured without or with paclitaxel (0.1–10 µM) for
24 h. Supernatants were collected and assayed for IL-12
heterodimer, as described. Paclitaxel (10 µM) significantly
(p < 0.05) enhanced both NH and TBH M IL-12
production (Fig. 3), increasing IL-12
levels by 64% and 112%, respectively. Paclitaxel doses as low as 0.1
µM enhanced NH M IL-12 production, although doses more than 10
µM did not further enhance NH or TBH M IL-12 production (not
shown). Splenic M cultures produced similar profiles, although total
IL-12 production by all samples was approximately 50% less than
comparably treated peritoneal M cultures (not shown). The induction
of IL-12 was not an artifact of endotoxin contamination because boiling
paclitaxel for 60 sec abrogated its capacity to induce IL-12 (not
shown).
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IL-12 p40
expression
Abrogation of TBH M p40 expression (see Fig. 2
A) and
paclitaxel-mediated reconstitution of TBH M IL-12 heterodimer
production (see Fig. 3) suggest that paclitaxel may induce IL-12 p40
expression. To test this hypothesis, NH or TBH peritoneal Ms (5
x 106 cells) were IFN- primed and cultured for 4 h
without or with various doses of paclitaxel (0.1–10 µM); p40
expression was assessed by RPA (Fig. 4).
Paclitaxel induced IL-12 p40 expression in a dose-dependent manner in
both NH (Fig. 4A) and TBH Ms (Fig. 4B).
Paclitaxel doses more than 10 µM did not further enhance p40
expression (not shown). Expression of GAPDH was consistent throughout
all samples.
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IL-12 production through NO
We (2, 3) and others (17) have shown that paclitaxel stimulates
M production of the pleiotropic effector molecule NO. Because NO
regulates IL-12 gene expression in the RAW264.7 M cell line (38), we
determined whether paclitaxel-induced NO may be involved in the
stimulation of murine M IL-12 production (Fig. 5). IFN--primed (10 U/ml) NH or TBH
peritoneal Ms were cultured for 24 h without or with paclitaxel
(10 µM) and the specific NO-inhibitor NMMA (0.1 mM, sufficient to
eliminate measurable NO production without compromising M
viability). Paclitaxel significantly (p <
0.05) enhanced NH M IL-12 production, but paclitaxel’s capacity to
induce IL-12 was blocked with the addition of NMMA. Tumor growth
negatively modulates but does not abrogate paclitaxel-induced M NO
production (3, 4). Paclitaxel significantly (p
< 0.05) enhanced TBH M IL-12 production, and paclitaxel-mediated
IL-12 production was NO-dependent (Fig. 5). In the absence of NO
production, paclitaxel-induced NH or TBH M IL-12 production was not
significantly different from basal levels, and NMMA alone did not
influence IL-12 production (Fig. 5).
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Tumor growth induces M suppressor activities (1), compromising
T cell proliferative responses in alloantigen-activated MLR cultures
(39, 40, 41). We reported that paclitaxel pretreatment of TBH Ms
partially reversed tumor-induced suppressor functions (3). Because
paclitaxel induces M IL-12 production and IL-12 can enhance T cell
proliferative responses (42, 43, 44), we assessed whether
paclitaxel-induced IL-12 was responsible for the reversal of TBH
M-mediated immune suppression by measuring lymphocyte responsiveness
in culture with paclitaxel-pretreated Ms in the absence or presence
of IL-12. NH BALB/c (H-2d) CD4+ T cell (2
x 105 cells) responsiveness to alloantigen stimulation
(4 x 105 X-irradiated C3H [H-2k]
splenocytes) was assessed in MLR with TBH Ms (2 x
105 cells). Ms were pretreated (4 h, optimal time)
without or with paclitaxel (10 µM) before addition to the MLR.
Alloreactivity was assessed in the absence or presence of neutralizing
anti-IL-12 mAb (C17.8, 10 µg/ml), anti-IL-2 (S4B6), or rat
IgG2a isotype control mAb (10 µg/ml). Data are expressed as magnitude
of response relative to proliferation in the presence of unactivated NH
Ms (control cpm was 86,460; not shown).
Paclitaxel pretreatment of TBH Ms partly reverses tumor-induced M
suppressor activity, allowing for significantly
(p < 0.005) enhanced alloantigen-activated T
cell proliferation (Fig. 6) in the
presence of TBH Ms. Neutralization of IL-12 activity in culture with
anti-IL-12 mAb did not significantly affect T cell proliferation in
the presence of untreated Ms. In contrast, neutralization of IL-12
abrogated paclitaxel-mediated reversal of tumor-induced suppression.
Neither anti-IL-2 mAb nor rat IgG2a isotype control affected
lymphocyte reactivity (not shown), suggesting that paclitaxel-induced
IL-12 is the principle factor responsible for reconstitution of T cell
reactivity.
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-derived NO is required for paclitaxel-induced IL-12
expression (see Fig. 5), we assessed whether paclitaxel-induced NO
production plays a role in the reversal of tumor-induced M
suppressor functions. CD4+ NH T cell alloantigen reactivity
was assessed, as described, in the absence or presence of the
NO-inhibitor NMMA (Fig. 7). NO
production, as measured by the Griess reaction, was 10.1 ± 0.3
µM and 37.4 ± 2.8 µM in cultures containing untreated or
paclitaxel-treated TBH Ms, respectively. NO production in cultures
containing NMMA was beyond the limit of detection of our assay system.
Paclitaxel pretreatment failed to relieve tumor-induced M suppressor
activity in the presence of the NO inhibitor, demonstrating that NO is
involved in the paclitaxel-mediated reversal of tumor-induced M
suppressor activity. Addition of exogenous IL-12 (1000 pg/ml,
approximately equal to paclitaxel-induced (10 µM) IL-12 production by
TBH Ms; see Fig. 3) to NMMA-containing cultures reconstituted T cell
proliferative response (Fig. 7). Data are expressed as magnitude of
response relative to proliferation in the presence of unactivated NH
Ms (control cpm was 80,710; not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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phenotype and function.
Much research has focused on defining how tumors induce M production
of immunomodulatory signals (reviewed in 1), demonstrating that
these factors are important mediators of immunosuppression during
cancer. However, our recent studies on the immune activities of the
anticancer drug paclitaxel led us to address tumor-induced immune
suppression from a different point of view. Perhaps, in addition to
overexpression of inhibitory cytokines, host antitumor response may be
compromised by the lack of a necessary priming, activating, or
costimulatory signal(s), preventing the induction of immune responses
to tumors. An ideal candidate for study was M-derived IL-12
(reviewed in 45), a novel heterodimeric cytokine that enhances
antitumor cytotoxic responses (46, 47), and a single factor that
possesses the potential to correct many TBH immune dysfunctions.
Pleiotropic in its activities, IL-12 induces T cell (48) and NK cell
(49, 50) IFN- production and promotes T cell development along the
Th1 pathway (51, 52, 53, 54). These functions led to speculation that IL-12
could be useful as an anticancer immunotherapeutic agent (55, 56), and
clinical trials using direct administration (23, 57) and gene therapy
approaches (24, 58, 59) have yielded promising results. The capacity of
exogenous IL-12 to partially restore TBH immunocompetence suggests that
tumor-induced M dysfunction may involve decreased production of
IL-12. Dysregulation of M IL-12 may occur among both tumor-proximal
and -distal immune cell populations if the tumor produces or induces
the production of immunomodulatory factors such as IL-10 and TGF-ß1
(60), as occurs in our tumor model (1, 30). Handel-Fernandez et al.
(61) reported that a nonmetastatic mammary adenocarcinoma (D1-DMBA-3)
dysregulated peritoneal exudate M p40 expression through
PGE2 and phosphatidyl serine. Using a p70-specific ELISA to
avoid the potential interference of excess free p40 or p40 homodimers,
we found that murine fibrosarcoma growth significantly
(p < 0.05) inhibited IFN--induced IL-12 p70
production by 74%. Although LPS enhanced IFN--primed TBH M
IL-12, the level of production remained significantly
(p < 0.005) less than similarly treated NH
Ms (Fig. 1). Most striking, optimal LPS triggering of TBH Ms
could not induce IL-12 production even to the levels of untriggered
IFN--primed NH Ms. Tumor-induced dysregulation of IL-12 occurs at
the level of transcription, since p40 expression was largely abrogated
in tumor-induced Ms regardless of priming or triggering with IFN-
and LPS, respectively (Fig. 2). Because IL-12 drives the induction of
the Th1-type immune response (the most effective antitumor response),
the dysregulation of IL-12 production could effectively disable this
arm of the immune response and allow tumors to grow unhindered.
Recognizing that tumor growth negatively regulates M production of
immunostimulatory factors, it followed that therapeutic agents that
reconstitute immune activity through indirect action could be inducing
the production of stimulatory cytokines. Pretreatment of TBH Ms with
the anticancer chemotherapeutic agent paclitaxel partially reversed
M-mediated suppressor activity (3). This suggested that paclitaxel,
which has demonstrated M-activating functions in vitro (2, 17) and
in vivo (3), may enhance T cell reactivity by activating M IL-12
production. Analyzing for IL-12 p70 showed that paclitaxel
significantly (p < 0.05) enhanced
IFN--primed M IL-12 production regardless of tumor growth (Fig. 3), and RPA analysis showed that paclitaxel activates p40 expression in
a dose-dependent manner (Fig. 4).
Although our data strongly suggest that paclitaxel induces IL-12
production and p40 expression, the mechanisms remained to be clarified.
One possibility involved paclitaxel-mediated induction of M NO
production. NO regulates IL-12 gene expression in the RAW264.7 M
cell line (38), and paclitaxel stimulates M production of NO (2, 3, 17). We determined that NO is an essential regulator of
paclitaxel-mediated IL-12 production (Fig. 5) in our model system.
Paclitaxel (10 µM) significantly (p < 0.05)
enhanced both NH and TBH M IL-12 production, but paclitaxel’s
capacity to induce IL-12 was substantially blocked with the abrogation
of NO production. These data suggest that a primary mechanism of IL-12
induction by paclitaxel may involve autocrine signaling through
M-derived NO.
Although tumors mediate functional and phenotypic changes in M
populations, T cells may not be functionally altered, opening the
possibility that restoration of M IL-12 production through
chemotherapeutics may be beneficial to the TBH. Paclitaxel pretreatment
of TBH Ms partially reversed tumor-induced suppressor functions (3),
and we determined that paclitaxel-mediated M IL-12 production was
responsible for the reversal of immune suppression (Fig. 6).
Neutralization of IL-12 activity in T cell alloreactivity cultures
abrogated paclitaxel-mediated reversal of tumor-induced suppression,
suggesting that paclitaxel’s apparent immunotherapeutic activity is
achieved through the induction of M IL-12 production. This suggests
that the TBH immune system may be fully capable of responding to cancer
if provided with appropriate stimulatory signals.
Interestingly, paclitaxel-induced M NO production plays a role in
the restoration of immunocompetence. In the presence of the NO
inhibitor NMMA, paclitaxel pretreatment failed to relieve tumor-induced
M suppressor activity (Fig. 7). These results provide an intriguing
link between IL-12 and NO, molecules with disparate function in tumor
immunity. In terms of signaling mechanisms, Schwacha and Eisenstein
(62) reported that IL-12 was necessary for the induction of NO
production in an infectious disease model, and Wigginton et al. (63)
showed that IL-12 primed TBH Ms for enhanced NO production. These
studies suggest that IL-12, through its capacity to induce IFN-
production, leads to priming of M populations for enhanced NO
production (IL-12 has no direct effect on M NO production; D.
W. Mullins and K. D. Elgert, unpublished observation). This report
suggests an additional regulatory pathway by demonstrating that NO can
induce M IL-12 production through an autocrine mechanism, thus
perpetuating the cycle of activation and enhancing the potential for
strong cell-mediated immune responses.
In terms of T cell reactivity, reconstitution of alloantigen-activated
T cell proliferative response in the presence of NO and loss of
response in the absence of NO production may seem counterintuitive. NO
is a potent antagonist of lymphocyte proliferative responses and can
severely dysregulate T cell reactivity (64, 65, 66). In our model system,
we observed that LPS enhances M NO production, leading to drastic
decreases in T cell alloreactivity, and blocking NO with specific
inhibitor relieves suppression. In the TBH, Ms are primed for
enhanced NO production on LPS signaling, but these same cells are
incapable of substantial IL-12 production (Fig. 1). When
LPS-activated TBH Ms are added to alloantigen-activated NH
CD4+ T cells, proliferation remains severely compromised,
partly because of high levels of M NO. In contrast, paclitaxel
pretreatment induces TBH M production of NO at lower levels, which
may have direct inhibitory effects of the T cells; however, that
M-derived NO also acts in an autocrine manner to induce M IL-12
production, which in turn enhances T cell proliferative responses (Fig. 6). Administration of exogenous IL-12 enhances IFN- in TBH (67),
which in turn can enhance M-inducible NO synthase expression and NO
production in situ, leading to an amplification of antitumor responses.
These results suggest that the restoration of IL-12 has significant
positive implications for the TBH.
Controversy surrounds human monocyte/M production of NO (68),
leading to questions about the relevancy of murine data to human
oncology studies. Human Ms do possess the gene and functional
protein for NO production (69), and NO production occurs in vitro (69, 70). Recent data indicate that human peripheral blood mononuclear cells
can produce moderate amounts of NO in vivo (70). From the opposing
view, it could be argued that lack of NO production by human Ms may
contribute to tumor-induced immunosuppression by the loss of IL-12 and
IFN- induction mechanisms. Regardless of human M NO production,
further investigation of M responsiveness to paclitaxel in human
cancer patients is warranted.
Collectively, these results demonstrate that the antineoplastic agent paclitaxel restores IL-12 production in the TBH, a novel immunotherapeutic activity that may help correct tumor-induced immune dysfunction. Further, these data ascribe a novel signaling component to the pleiotropic activities of NO. We suggest that the immune activities of paclitaxel should be considered in a clinical context, because recognition of paclitaxel’s immune-activating properties may lead to optimization of current chemotherapeutic treatment regimens.
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Acknowledgments |
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Footnotes |
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2 Current address: Beirne B. Carter Center for Immunology Research, MR4 Box 4012, University of Virginia Health Sciences Center, Charlottesville, VA 22908.
3 Address correspondence and reprint requests to Dr. Klaus D. Elgert, Microbiology and Immunology Section, Department of Biology, Virginia Polytechnic Institute and State University, 2119 Derring Hall, Blacksburg, VA 24061-0406. E-mail address:
4 Abbreviations used in this paper: M, macrophage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NMMA, NG-monomethyl-L-arginine; NH, normal host; RPA, ribonuclease protection assay; TBH, tumor-bearing host.
Received for publication November 20, 1998. Accepted for publication March 19, 1999.
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