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,
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Dipartimento di Oncologia e Neuroscienze, Università di Chieti, Chieti, Italy;
Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy;
Dipartimento di Oncologia Clinica e Sperimentale, Università di Genova, Genova, Italy; and
§
Institute of Medical Virology University of Zurich, Zurich, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
IFN-
, and at higher levels GM-CSF, macrophage-inflammatory
protein-2, and monocyte chemoattractant protein-1. In NK cell-depleted
nude mice, numerous macrophages and granulocytes infiltrated the tumor,
and a strong expression of macrophage-inflammatory protein-2 and
inducible NO synthase was also observed. Finally, macrophages
cocultured with N592/IL-12/IL-15 produced NO in vitro, and inhibited
tumor cell growth, further suggesting their role as effector cells in
this model. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among natural immunity effectors, NK cells are known to recognize and lyse cells that lack expression of MHC class I (13, 14). In fact, the expression of MHC class I on normal cells confers protection from NK cell lysis, through NK-inhibitory signals mediated by killer-inhibitory receptors for MHC class I (15, 16). Thus, down-regulation of MHC class I expression in tumor cells results in enhanced NK susceptibility.
IL-15 is a four -helix bundle cytokine displaying IL-2-like
immunostimulatory functions (17, 18), supporting the
proliferation and differentiation of T, B, and NK cells. In addition,
IL-15 has been reported to control differentiation of NK cells from
bone marrow precursors (19), to stimulate NK antitumor
cytolytic functions (20, 21), and to act as a chemotactic
stimulus for NK cells (22). The critical role of IL-15 in
NK cell development and function was also evidenced in IFN-regulatory
factor 1 (IRF-1) knockout mice, which lack IL-15 expression and display
an NK-deficient phenotype (23). In view of these
properties, IL-15 has been regarded as a suitable candidate for cancer
immunotherapy (24) or gene therapy strategies (25, 26).
In a previous study, we have shown that a human MHC class I-negative tumor, engineered to secrete IL-15, displayed a reduced growth and take rate when xenotransplanted in nude mice, although tumorigenicity was not completely abrogated (27). High number of infiltrating NK cells were found at the tumor site, a finding that has been rarely observed in cytokine-transduced tumors (28), and the IL-15-mediated effects were abrogated by NK cell depletion. We speculated that the combined gene transfer of IL-15 with other NK-stimulating factor(s) could synergistically enhance the antitumor effects of NK cells recruited by IL-15 at the tumor site.
IL-12 is a heterodimeric cytokine, secreted by monocytes, macrophages,
and dendritic cells, which is able to activate both T and NK cell
functions. Thus, IL-12 is a potent inducer of Th1 responses and induces
NK cell proliferation, cytotoxic activity, and IFN- production
(29). Several studies have demonstrated a potent antitumor
activity of IL-12 either as a recombinant cytokine or in gene transfer
approaches in different syngeneic mice models (29, 30, 31).
Another report showed that the combination of IL-12 and IL-15
synergistically potentiated cytokine production by NK cells in vitro
(32). In addition, a combination of suboptimal doses of
the two recombinant cytokines induced antitumor effects in a B16
melanoma model in syngeneic mice (33).
In this study, we have analyzed the possible cooperative antitumor effects of IL-12 and IL-15 gene transfer in a human small cell lung cancer cell line, N592, chosen as a prototype of MHC class I-negative tumor. The effects on natural immunity were studied by xenotransplantation in nude mice. Interestingly, engineering of tumor cells with both IL-15 and IL-12 resulted in a complete tumor rejection also in NK-depleted or in granulocyte-depleted nude mice, suggesting a predominant role of macrophages in this model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N592 small cell lung cancer cell line was kindly provided by Dr. J. Minna, National Cancer Institute (Washington, D.C.). Cells were cultured in endotoxin-free RPMI 1640 medium (endotoxin content <0.005 EU/ml) supplemented with L-glutamine and antibiotics (all from BioWhittaker, Bergamo, Italy) and 10% FCS (endotoxin content 20 EU/ml; Seromed Biochrom, Berlin, Germany).
Plasmid vector assembly and N592 transfection
The pVkL/IL-15IRESneo plasmid vector was obtained as previously described (27, 34). The bicistronic mIL-12 cDNA, encoding for p40 and p35 murine IL-12 chains, was digested from pIRES-muIL-12 (35) with BamHI and was subcloned in pIRES1neo or pIRES1hygro plasmid vectors (Clontech Laboratories, Palo Alto, CA). The orientation of the insert was checked by further digestion with BstXI restriction enzyme.
N592 cells were transfected with 10 µg of pVkL/IL-15IRES1neo or pmuIL-12IRES1hygro plasmids or both using cationic liposomes (DOTAP; Boehringer/Roche, Milano, Italy).
Stable transfectants and clones were obtained by limiting dilution, in medium containing either G418 (500 µg/ml) or hygromicin (250 µg/ml) or both, and were then tested for IL-15 and IL-12 production.
IL-15, IL-12, GM-CSF, and IFN- ELISA or bioassays
As indicator cell system for determination of IL-2/IL-15 activity, we used the CTLL mouse cell line, known to proliferate in response to human IL-2 or IL-15. Cytokine activity was assessed by [3H]thymidine uptake by CTLL after 6-h pulse with 0.5 µCi at the end of 24-h period of incubation with supernatants of cell lines or of transfectants. Serial dilutions of human rIL-2 or human rIL-15 (Genzyme, Cambridge, MA) containing a known amount of IU were used as standard.
IL-15 or IL-12 ELISA was performed using a commercial available kit according to instructions provided by the manufacturer (Genzyme).
The production of murine IFN- and GM-CSF by nude mice splenocytes
cocultured with N592pc or transfectants was analyzed by an ELISA kit
(Genzyme) on 7-day supernatants.
Nude mice studies
Pathogen-free female athymic (nu/nu, CD1) mice, 6–8 wk old, were obtained from Harlan Nossan (Milano, Italy). Mice were housed under pathogen-free conditions and received autoclaved food and water.
Animals (six mice for each group) were injected s.c. with 2 x 107 N592pc, N592/neo/hygro, N592/IL-12, N592/IL-15, or N592/IL-12/IL-15 tumor cells. Cells were mycoplasma free, as assessed by ELISA (Boehringer/Roche) or 4',6'-diamidino-2-phenylindole staining before injection. Cells were washed three times in endotoxin-free RPMI medium without FCS and one time in endotoxin-free PBS before injection. The larger and smaller diameters of the s.c. tumors were measured using a caliper at weekly intervals; these two diameters were multiplied to obtain an estimate of the tumor area. The data are displayed as mean ± SD of the areas for each group of animals at a given time point. Statistical analysis was performed using the Mann-Whitney test; p < 0.05 values were considered as significant.
NK or granulocyte depletion was performed by i.p. injection of rabbit anti-asialo GM1 antiserum (Wako Chemicals GmbH, Dussendorf, Germany) (0.2 ml of 1/20 diluted stock solution) or anti-granulocyte rat mAb (0.2 ml of a 1/50 dilution of ascites of RB6-8C5 hybridoma; kindly provided by Dr. R. L. Coffman, DNAX, Palo Alto, CA). Mice were injected at days -2, 0, +5, +10, and +17 from tumor challenge. Efficiency of depletion (>80% in case of granulocytes and >95% in case of NK cells) was confirmed by immunohistochemical analyses of the infiltrate of xenotransplanted N592/IL-12/IL-15 tumors. Control animals received normal rabbit serum or an irrelevant rat mAb.
Morphologic and immunohistochemical analysis of xenografts
Groups of three mice were euthanized 5 and 7 days after challenge. For histologic evaluation, tissues were fixed in 10% neutral buffered Formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin-eosin or Giemsa.
For immunohistochemistry, acetone-fixed cryostat sections were
incubated for 30 min with anti-asialo GM1 (NK cells) (Wako
Chemicals GmbH); anti-Mac-1 (anti-CD11b/CD18), anti-Mac-3,
and anti-Ia (all from Boehringer Mannheim);
anti-polymorphonuclear leukocytes (RB6-8C5 hybridoma; provided by
Dr. R. L. Coffman, (DNAX)); anti-endothelial cells
(mEC-13.324) and anti-endothelial leukocyte adhesion molecule 1
(anti-ELAM-1) (provided by Dr. A. Vecchi, Negri Nord, Milano, Italy);
anti-IL-1ß and anti-GM-CSF (Genzyme); anti-TNF-
(Immuno Kontact, Frankfurt, Germany); anti-IFN- (provided by Dr.
S. Landolfo, University of Turin, Turin, Italy); anti-monocyte
chemoattractant protein-1
(anti-MCP-1)3 and
anti-VCAM-1 (PharMingen); anti-macrophage-inflammatory protein-2
(anti-MIP-2) (Serotec, Oxford, U.K.); anti-ICAM-1 (Santa Cruz
Biotechnology, Santa Cruz, CA); and anti-inducible NO synthase
(anti-iNOS) (Transduction Laboratories, Lexington, KY) Abs. After
washing, they were overlaid with biotinylated goat anti-rat,
anti-hamster, and anti-rabbit, and horse anti-goat Ig
(Vector Laboratories, Burlingame, CA) for 30 min. Unbound Ig was
removed by washing, and the slides were incubated with ABC (avidin
biotin complex) alkaline phosphatase (Dako, Glostrup, Denmark).
Quantitative studies of immunohistochemically stained sections were
performed independently by three pathologists in a blind fashion. For
cell counts, individual cells were counted under a microscope x400
field (x40 objective and x10 ocular lens; 0.180
mm2 per field). Ten randomly chosen fields were
counted in each sample. The expression of cytokines and iNOS was
defined as absent (-), scarcely (±), moderately (+), frequently (++),
and strongly (+++) present on cryostat sections tested with the
corresponding Abs.
Isolation of granulocyte and macrophage populations, production of NO, and cytotoxicity
To isolate granulocytes, nude mice were injected i.p. with 1 ml of 9% sodium casein (Sigma-Aldrich, Milano, Italy) in endotoxin-free PBS, followed by a second injection 16 h later. Three hours later, peritoneal cells were recovered in 5 ml of DMEM containing 0.5 mM EDTA. To isolate macrophages, nude mice were injected i.p. with 1 ml of 2.9% aged thioglycolate (Difco, Detroit, MI) solution. After 2 days, peritoneal cells were harvested as above. Cell populations were fractioned on self-forming 90% Percoll (Pharmacia Biotech, Uppsala, Sweden) gradients by centrifugation at 60,000 x g for 20 min at 4°C, which produced a two-layer fractionation pattern. Macrophages were harvested from the upper layer of thioglycolate-induced peritoneal cell populations, while granulocytes were collected from the lower layer of casein-induced cells. Purity of each cell population was >90%, as judged by microscope examination of stained cytospin preparations.
Macrophages or granulocytes were cocultured with different N592 transfectants (2 x 104 in 1 ml of medium) at 10:1 or 20:1 ratios in 24-well plates. NO production in the supernatant was measured after 2 or 5 days by a colorimetric assay using the Griess reagent (Sigma-Aldrich) (36). Cytotoxicity against N592 transfectants, which grow in suspension, was evaluated by gently resuspending N592 and transferring them in triplicate wells of a 96-well flat-bottom plate. The amount of viable cells was evaluated by a standard MTT (Sigma-Aldrich) assay (37).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N592 cells were transfected with pVkL/IL-15-IRESneo and/or
pIL-12-IREShygro, either alone or in combination, and cloned after
appropriate drug selection. The modified IL-15 cDNA
VKL/IL-15 encodes for an IL-15 preprotein bearing the Ig
L chain signal peptide that allows enhanced secretion of biologically
active IL-15, upon transfection, as compared with unmodified IL-15
cDNAs (34). The N592 clones listed in Table I
were selected for further studies
because they displayed similar growth kinetics in vitro and on the
basis of their cytokine secretion pattern. Thus, the N592/IL-12/IL-15
clone secreted amounts of IL-15 or IL-12 similar to that produced by
the clones expressing only IL-15 or IL-12. IL-15 secreted by
transfectants was biologically active in sustaining both
IL-2/IL-15-sensitive CTL-L proliferation and in boosting NK cytolytic
activity (27).
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The effect of cytokine engineering on tumorigenicity was evaluated
by heterotopic (s.c.) implant in nude mice possessing a functional
natural immunity. As shown in Fig. 1A, mock-transfected N592
(N592/neo/hygro) showed a very rapid growth kinetics in 100% of
injected animals, which was similar to that of unmodified N592.
N592/IL-12 cells displayed only minor changes of the growth pattern,
while N592/IL-15 showed a clearly reduced tumor growth rate and a
slight reduction in tumorigenicity (80% tumor take). However,
N592/IL-12/IL-15 produced only a transient tumor growth, followed by
complete rejection in all animals tested. Consistent results were
obtained in three unrelated experiments.
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B). Thus, a very potent local bystander effect on N592pc
was produced by s.c. injection of N592/IL-12/IL-15. The weak systemic
bystander effect, observed in animals injected with N592pc
contralaterally to N592/IL-12/IL-15, may be related to the low levels
of circulating IL-15 (15 pg/ml at day +3, undetectable at day +7). In
contrast, consistent levels of IL-12 (1600 pg/ml) were found at day +3,
and it was still detectable at day +7. Effect of NK cell and granulocyte depletion on N592 transfectant tumor growth
To gain further information on the role of NK cells, N592/IL-15
and N592/IL-12/IL-15 transfectants were injected in nude mice that had
been treated with anti-asialo GM1 antiserum. N592/IL-15 and
N592/IL-12 showed growth kinetics similar to that of N592pc in
NK-depleted nude mice, while N592/IL-12/IL-15 were still completely
rejected (Fig. 2). Treatment with
nonimmune rabbit control Ig had virtually no effect (Fig. 2B). These findings indicate that NK cells are mainly
responsible for the antitumor effect of IL-15, while these cells are
not necessary for the IL-15/IL-12 cooperative effect. In addition,
N592/IL-12/IL-15 were also rejected in granulocyte-depleted nude mice,
although a delay in the rejection process was observed in these animals
(Fig. 2C). Similar results were observed in nude mice
depleted of both NK cells and granulocytes (data not shown), thus
suggesting that macrophages play a predominant role.
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and GM-CSF
secretion by murine splenocytes in vitro
We further analyzed whether IL-15 and IL-12 produced by
transfectants could synergize in inducing cytokine production by mouse
splenocytes in a coculture system. As shown in Fig. 3A, after 3 days of coculture
with N592/IL-12/IL-15, splenocytes released 1500 pg/ml of IFN- in
the supernatant, vs average levels of 10 and 110 pg/ml measured in
N592/IL-15 and N592/IL-12 coculture supernatants, respectively. Similar
results were obtained for GM-CSF (Fig. 3B). Thus, 15 and 50
pg/ml of GM-CSF were found in cocultures with N592/IL-15 and
N592/IL-12, respectively, while 100 pg/ml was secreted in
N592/IL-12/IL-15 cocultures. This cooperative effect of IL-15 and IL-12
was largely dependent on NK cell stimulation because splenocytes from
anti-asialo GM1-depleted animals produced lower levels of IFN-
or GM-CSF (Fig. 3, A and B), as compared with
splenocytes from untreated animals or from animals treated with control
rabbit Ig (not shown).
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To gain further information on the mechanisms underlying the cooperative effects of transfected IL-15 and IL-12 in vivo, we performed histological and immunohistochemical analysis of the N592pc, N592/IL-12, N592/IL-15, and N592/IL-12/IL-15 tumor area in intact or NK-depleted nude mice.
When injected in nude mice, either N592/pc or N592/neo/hygro cells gave
rise to a richly vascularized tumor arranged in small alveolar
structures with very few infiltrating macrophages (Fig. 4a). A similar growth pattern
was observed in N592/IL-12 tumor growth area in which, however,
microvessels were less numerous and a few necrotic areas were present
(Fig. 4d). The periphery of the tumor mass was moderately
infiltrated by reactive cells producing proinflammatory cytokines and
expressing iNOS (Table II). Production of
granulocyte chemotactic mediators, such as GM-CSF and particularly
MIP-2, was detectable in tumor-infiltrating macrophages.
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g),
numerous but severely damaged microvessels, and several
infiltrating reactive cells, particularly macrophages and NK cells
(Fig. 4h). A weak expression of GM-CSF and a strong
endothelial and macrophage expression of MCP-1 were detected, while
proinflammatory cytokines were almost absent (Table II).
N592/IL-12/IL-15 tumor rejection area mainly consisted in a wide area
of ischemic necrosis with colliquative foci and small groups of ghosts
of tumor cells interspersed within numerous macrophages, granulocytes,
and NK cells (Fig. 4, j–l, and Table II). Vascularization
was scarce to absent with diffusely damaged microvessel sprouts.
Production of proinflammatory cytokines was quite similar to that
observed in N592/IL-12 tumor growth area, while production of GM-CSF,
MIP-2, and MCP-1 was stronger.
In NK cell-depleted nude mice, N592/IL-12/IL-15 tumor rejection area
was markedly infiltrated by macrophages and granulocytes with a
moderate production of IL-1ß, TNF-, and MCP-1, and a strong and
widely distributed expression of iNOS and MIP-2 (Fig. 5, a–e, and Table II).
Anti-endothelial cell (anti-CD31) staining was scarce to absent
with a dusty appearance, indicating the heavily injured endothelial
cells (Fig. 5f).
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In the attempt to identify the cells responsible for NO
production, we first isolated thioglycolate-induced macrophages or
casein-elicited granulocyte fractions by density gradients and tested
their ability to produce NO in coculture with N592/IL-12/IL-15. As
shown in Fig. 6A, only
macrophage-enriched populations released significant amounts of NO in
response to coculture with N592/IL-12/IL-15, while granulocyte-enriched
fractions did not (Fig. 6A). When macrophages and
granulocytes were cocultured together with N592/IL-12/IL-15, a slight
cooperative effect in NO production was observed.
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B). In addition, macrophage-enriched populations markedly
inhibited N592/IL-12/IL-15 growth in a 5-day MTT assay, while N592pc
were not affected, and N592/IL-12 and N592/IL-15 were only partially
inhibited (Fig. 6C). The finding that N592/IL-15 growth was
partially inhibited by macrophages, in the absence of NO production,
suggested that both NO-dependent and NO-independent mechanisms of tumor
cell growth inhibition were operative. This possibility was also
suggested by the use of the iNOS inhibitor
L-NAME, (N
-nitro-L-arginine methyl
ester) which potently inhibited NO production by macrophages at 5 mM,
but only partially blocked cytotoxicity (Fig. 6D) against
N592/IL-12/IL-15. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although IL-12 has been proven to exert antitumor effects in a number
of experimentally induced and spontaneous tumors in immunocompetent
hosts (29, 30, 31), it had virtually no efficacy in our tumor
gene transfer model in nude mice. In the absence of T lymphocytes,
N592/IL-12 tumor growth kinetic was not affected despite an evident
aspecific reactive cell infiltration and proinflammatory cytokine
production at the tumor site. Furthermore, small sprouting vessels,
mainly found at the periphery of tumor mass, may support tumoral cell
survival, making easier tumor escape from any attempt of host natural
immune response. Altogether, these observations suggest the requirement
of T cells in leading to an efficient IL-12-induced antitumor reaction.
Furthermore, a pivotal role of CD8+ cells has
been found in the rejection of IL-12 gene-transfected cells injected in
syngeneic mice (31). The involvement of
CD8+ cells was frequently associated with an
indirect angiogenesis inhibition by secondary cytokines (mainly
IFN-) and third-level chemokines (inflammatory protein-10 and
monokine induced by IFN- (MIG)) released by CD8+
themselves or induced in other leukocyte subsets (29).
Hence, from here, it may be hypothesized that the failure of IL-12
immunotherapy in several clinical trials could be ascribed, besides to
the advanced stage of cancer patients (bearing established and
widespread tumors), to an inadequate T cell responsiveness against
weakly immunogenic and well-vascularized tumors.
Although two isoforms of IL-15 mRNA are constitutively expressed in
human tumors (34, 38), spontaneous secretion of IL-15 by
tumor cells was not observed due to the existence of multiple
posttranscriptional levels of IL-15 production control (18, 39). We previously reported that the use of a modified human
IL-15 cDNA for gene transfer allowed secretion of IL-15 that was found
to be biologically active on both human and murine NK and T cells
(34). Injection of IL-15-engineered N592 tumor cells in
nude mice resulted in an NK cell-dependent inhibition of tumor growth.
In the light of the synergistic or additive effects exerted by low
doses of IL-12 and IL-15 on NK and lymphokine-activated killer
cytotoxicities (40), with the reciprocal up-regulation of
receptor expression on mononuclear cells (33, 41), one may
expect an NK cell-mediated rejection of N592 cells releasing both
cytokines. In addition, it has been reported that IL-15 synergistically
potentiates IL-12-induced NK cell production of several cytokines such
as IFN-, IL-10, MIP-1, and MIP-1ß (32). Moreover,
a cooperative antitumor effect, induced by subtherapeutic doses of
recombinant IL-12 + IL-15 in a B16F10 melanoma model in syngeneic mice,
correlated with the level of IFN- production (33). In
agreement with these previous observations, nude mice splenocytes
cocultured with N592 secreting both IL-15 and IL-12 released higher
amounts of IFN- and GM-CSF than cocultures with N592 transfectants
producing only IL-12 or IL-15. Conversely, cytokine production was
clearly reduced in coculture of splenocytes isolated from NK-depleted
animals, indicating that also in nude mice NK cells were mainly
responsible for IL-12 + IL-15-induced IFN- and GM-CSF production.
However, NK cell depletion of nude mice still resulted in a complete
N592/IL-12/IL-15 tumor cell rejection in vivo, thus suggesting the
involvement of other effector cells of natural immunity in IL-12 +
IL-15-induced antitumor reaction.
Immunohistological analysis of N592/IL-12/IL-15 tumor area revealed a marked macrophage and granulocyte recruitment, associated with a strong iNOS activation and large colliquative necrotic areas. It has been reported that the cytostatic/cytotoxic effect of ex vivo macrophages from mice treated with IL-12 and/or IL-15 was dependent, at least in part, on NO production (33). Our data indicate that nude mice macrophage populations cocultured with N592/IL-12/IL-15 produced NO and inhibited the growth of these tumor cells in vitro, through both NO-dependent and NO-independent mechanisms.
In contrast, granulocytes, attracted at the tumor site by MIP-2 (murine equivalent of IL-8) (42), may also cooperate in NO production (42, 43, 44, 45), and in tumor destruction (46, 47, 48, 49, 50). Both granulocytes and macrophages could also be responsible for the blood vessel injuries observed in N592/IL-12/IL-15 tumor (29, 50). In addition, it should be outlined that human neutrophils can respond to IL-12 stimulation (51) and can also be activated by IL-15 to express IL-8 (52). Indeed, granulocytes appeared to play a cooperative role in our model because in granulocyte-depleted animals, the N592/IL-12/IL-15 rejection process was delayed.
In NK cell-depleted nude mice, it is likely that the cooperative
biological effects of IL-12 and IL-15 mainly target macrophages and
granulocytes, allowing the onset of a distinct natural immunity
regulatory network. In this context, an increased iNOS and MIP-2
expression, detected at the tumor site, should be viewed as signs of an
increased IL-12 and/or IL-15 direct macrophage and granulocyte
activation (51, 52, 53, 54). Although NK-depleted nude mice showed
a reduced IFN- production in response to IL-12 + IL-15 produced by
transfectants, both in vitro and in vivo, it is likely that low amounts
of macrophage-derived IFN- (53) may play a role,
particularly in iNOS induction. Thus, low amounts of IFN- were found
in cocultures of peritoneal macrophages with double-transfected cells
(data not shown).
Different from previous studies in tumor cytokine immuno/gene therapy, which emphasize the requirement of a cross-talk between specific and nonspecific immune mechanisms to obtain a complete tumor rejection (55, 56), in this study we report evidence that the activation of macrophages by the synergistic action of IL-12 + IL-15 may lead to an effective T- and NK-independent tumor growth inhibition. These data provide new insight on the cooperative activity of IL-12 and IL-15 in the stimulation of effective natural immunity response and could be relevant for the development of gene therapy strategies against MHC class I-negative tumors escaping from specific CTL control.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Silvano Ferrini, Centro di Biotecnologie Avanzate, Largo Rosanna Benzi no. 10, 16132 Genova Italy.
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; iNOS, inducible NO synthase; L-NAME, N-nitro-L-arginine methyl ester; MIP, macrophage-inflammatory protein.
Received for publication December 16, 1999. Accepted for publication June 21, 2000.
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G. Gri, C. Chiodoni, E. Gallo, A. Stoppacciaro, F. Y. Liew, and M. P. Colombo Antitumor Effect of Interleukin (IL)-12 in the Absence of Endogenous IFN-{gamma}: A Role for Intrinsic Tumor Immunogenicity and IL-15 Cancer Res., August 1, 2002; 62(15): 4390 - 4397. [Abstract] [Full Text] [PDF]
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E. Bulanova, V. Budagian, T. Pohl, H. Krause, H. Durkop, R. Paus, and S. Bulfone-Paus The IL-15R{alpha} Chain Signals Through Association with Syk in Human B Cells J. Immunol., December 1, 2001; 167(11): 6292 - 6302. [Abstract] [Full Text] [PDF]
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 E. Di Carlo, G. Forni, P. Lollini, M. P. Colombo, A. Modesti, and P. Musiani The intriguing role of polymorphonuclear neutrophils in antitumor reactions Blood, January 15, 2001; 97(2): 339 - 345. [Full Text] [PDF]
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) or 20:1 (
) E:T cell ratio. B,
Production of NO by macrophage populations incubated with N592pc or
with different N592 transfectants at a 10:1 (