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Departments of Immunology and Pathology, Hospital Clínico San Carlos, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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plus CD40 ligation)
able to inhibit tumor cell growth in vitro. Early myeloid cells
(ER-MP54+) and cells expressing inducible NO synthase are
increased at the site of tumor challenge in mice treated with the
combined therapy, but not in those treated with Cy or immune cell
transfer alone. Thus, Cy induces the expansion of early myeloid cells,
inhibiting tumor cell growth by a mechanism involving NO. Both the
recruitment and the activation of these myeloid cells at the site of
tumor challenge appear to be dependent on the presence of
tumor-specific lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown that Cy induces the development of suppressor
cells in the spleens of mice 1–2 wk after drug administration
(10, 11, 12). These cells appear to be related to those
wheat-germ agglutinin (WGA)-positive cells described in normal bone
marrow mediating natural suppressor activity (13). This
activity is also found in the spleens of mice undergoing graft-vs-host
reactions (14), after total lymphoid irradiation
(15), or in mice bearing advanced tumors
(16). Natural suppression is defined functionally because
of its strong inhibition of lymphoproliferative responses
(13, 14, 15, 16). However, it may also inhibit the proliferation
of other cell types, including those of tumor origin (17, 18). In most instances this suppressor activity is
nonconstitutive, but is induced upon proper cell activation
(19). In this regard T cell signals have been described to
play a critical role (20, 21). Previously, we have shown
that bone marrow-derived natural suppressor activity is mediated by NO
(22), which is produced in large quantities by early
myeloid cells following IFN- plus CD40 stimulation
(23). Similar cells have been described recently to be
expanded in Cy-treated mice (24). The potent tumoricidal
activity of NO (25) makes it conceivable that an antitumor
effect of early myeloid cells developed upon Cy treatment might occur,
provided that proper T cell signals are given. Production of NO in
Cy-treated animals is supported, because this agent has been involved
in the pathogenesis of conditions associated with Cy treatment
(26, 27, 28).
Here we have assessed the effect of Cy plus tumor-specific lymphoid cell transfer on the tumorigenesis of a mouse mammary carcinoma (Ehrlich tumor (ET)). The results show that mice subjected to this combined therapy become resistant to ET cell challenge by a mechanism involving NO production. This production is associated with the development of early myeloid cells, which are located at the site of tumor challenge in these mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female F1 (C57BL/6J x DBA/2) mice (8–10 wk old) were obtained from the Experimental Unit, Hospital Clínico San Carlos (Madrid, Spain).
Reagents
Cy, WGA-FITC, streptavidin-FITC, streptavidin-PE, and mouse
rIFN- (<0.1 ng/µg endotoxin) were purchased from Sigma (St.
Louis, MO). Percoll was obtained from Pharmacia (Uppsala, Sweden).
NG-monomethyl-L-arginine
monoacetate salt (LMMA) was obtained from Calbiochem (San Diego, CA).
S-nitroso-N-acetyl-D,L-penicilamine
(SNAP) was purchased from Alexis (Laufelfingen, Sweden).
Antibodies
Mouse Ly-6C (FITC-conjugated rat IgG2a, ER-MP20 mAb) staining the cell surface of murine midstage macrophage precursor cells (29), CD31 (PECAM-1, biotinylated rat IgG2a, ER-MP12 mAb) staining the cell surface of murine early stage myeloid precursor cells (29), and ER-MP54 (rat IgG1 mAb) staining the cytoplasm of murine early stage myeloid precursor cells (29) from BMA (Augst, Switzerland) were used. FITC-conjugated rat anti-mouse Ly-6G (GR-1) mAb, purified rat anti-mouse CD16/CD32 mAb (Fc blocking), and biotinylated isotypic control mAbs (rat IgG2a, rat IgG1) from Caltag (San Francisco, CA) were also used. Mouse CD40 agonistic mAb (30) (purified rat IgG2a, 1C10; <4 endotoxin units/mg endotoxin contamination) was provided by A. W. Heath (Sheffield, U.K.). Rabbit anti-mouse inducible NO synthase (iNOS) was supplied by Alexis.
Ehrlich tumor
The hyperdiploid strain of ET was used. ET cells growing exponentially were collected from culture flasks and washed twice with HBSS, and viable cells (trypan blue dye exclusion) were counted. Cells were inoculated i.m. (5 x 105) in the left groin or, eventually, in the footpad of mice as indicated. In some experiments 5 mg of LMMA was administered locally in 25 µl at the site of tumor inoculation. This was done the day before tumor challenge and 10 days thereafter daily to inhibit NO production in vivo (31). Tumor size was estimated by calculating the product of two perpendicular diameters measured with Vernier calipers.
Preparation of immune and control spleen cells
Mice were immunized with three i.p. inoculations of irradiated (30 Gy) ET cells (107) once a week and used 10 days after the last dose. Spleen cells from immunized or control mice were prepared by gently pressing aseptically removed spleen through a 60-mesh stainless steel screen. Red cells were lysed with distilled water, and after a washing step, cell viability (>90%) was determined by trypan blue dye exclusion.
Cy treatment and immune cell transfer
Mice were treated with Cy and transferred with immune spleen cells following the protocol described by Proietti et al. (7) with slight modifications. Briefly, Cy was reconstituted with sterile PBS and injected i.p. (0.5 ml) to mice at 200 mg/kg (day 0). Five hours later, a cell suspension (0.1 ml) containing 108 immune or control spleen cells plus 2 x 105 irradiated ET cells was injected i.v. This cell transfer was repeated 6 days later. On day 7 mice were challenged (i.m.) with viable ET cells. In some experiments immune cell transfer was conducted as a Winn’s assay. Thus, control or immune spleen cells (5 x 106) were mixed with viable ET cells (5 x 105) in 0.1 ml and inoculated to mice treated, or not, with Cy 7 days before.
Preparation of Percoll spleen fraction 3 (Fr3) cells
Fr3 spleen cells were obtained from spleens of Cy-treated or control mice exactly as described previously (16). Briefly, plastic nonadherent cells obtained after overnight incubation were fractionated in discontinuous Percoll density gradients. The low density cell fraction banding between 50 and 60% of Percoll was referred to as Fr3, and the other cell fractions banding between higher densities (60–70 and 70–100%) were referred to as Fr2 and Fr1, respectively.
Flow cytometry
Immunofluorescence was performed using 200 x 103 spleen cells (erythrocyte free). Cells were preincubated in 50 µl of PBS, 0.1% azide, and 25% normal rat serum (30 min at 4°C). A saturating amount of labeled mAb or adequate isotypic controls were added at a final concentration of 10 µg/ml to the samples to be tested. Cells were incubated (30 min at 4°C) in the dark and washed. If necessary (e.g., when using biotinylated mAbs), cells were incubated with FITC- or PE-conjugated streptavidin for an additional 20 min. After a final washing step, the cells were fixed in PBS/1% paraformaldehyde and assayed in a flow cytometer (EPICS XL; Coulter, Miami, FL). In some experiments the cells were stained intracytoplasmatically with ER-MP54 mAb using a cell permeabilization kit (Fix & Perm; Caltag, Burlingame, CA). In all cases nonspecific staining was controlled by isotypic-matched Abs, and this background was subtracted from results.
Cell cultures
Cell cultures were performed with spleen cells derived from
Cy-treated mice to assess NO production under different stimuli.
Cultures were conducted in triplicate in 96-well flat-bottom cell
culture plates (Costar, Cambridge, MA) by seeding 200 x
103 cells/well in 250 µl of culture medium
(RPMI 1640, 10% FCS, 2 mM L-glutamine, 5 x
10-5 M 2-ME, and 80 µg/ml gentamicin). The
stimuli used were 1C10 (4 µg/ml) with or without IFN- (100 u/ml)
added to cultures on day 0. In some experiments proliferation of ET
cells (15 x 103/well) was tested in
cocultures with spleen cells (20 x
104/well) by measuring thymidine uptake in 48-h
cultures as previously described (32). In these
experiments the effect of NO on ET cell proliferation was assessed by
adding LMMA (0.5 mM), an inhibitor of NO synthesis (22).
Proliferation assays with ET cells were also performed in the presence
of different concentrations of SNAP, an NO donor agent, added at the
start of culture.
Measurement of NO production
NO production was estimated by the Griess reaction, measuring nitrite accumulation in 48-h culture supernatants as previously described (22). In proliferation assays supernatants were recovered before cell harvesting.
Histological studies
Histological studies were performed with hematoxylin-eosin and immunoperoxidase as previously described (33), using ER-MP54 or anti-iNOS on frozen sections obtained 2 or 6 days after tumor challenge. Quantitative studies of the immunohistochemically stained tissue sections were performed by an external pathologist in a fully blind manner on replicates of three or more coded samples. Cell counting was conducted using an image analyzer system (Q 500IW; Leica, Cambridge, U.K.) on 22 x 103 µm2.
Statistics
Data were analyzed using Microsoft Excel (Seattle, WA). Experimental differences from the controls were assessed by Student’s t test. p > 0.05 was considered nonsignificant. The experiments were conducted with groups of six mice per treatment, and at least three independent experiments were conducted to assure that the results were representative.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ET growth was assessed in mice treated with Cy 7 days before ET
challenge, with or without transfer of immune spleen cells. As shown in
Fig. 1
, Cy treatment did not inhibit ET
development in mice transferred with control spleen cells. Control mice
(Cy-untreated) that were transferred with immune cells systemically
(i.v.) (Fig. 1A), or locally as a Winn’s assay (Fig. 1B) increased their resistance against ET growth. As shown,
in each case this resistance was enhanced significantly if mice were
treated additionally with Cy. These results indicated that both Cy
treatment and immune cells are required for successful ET
suppression.
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plus CD40 ligation
Immature myeloid cells showing natural suppressor activity are
found in the spleens of mice treated with Cy (34). As bone
marrow-derived natural suppressor activity is mediated by NO produced
by early myeloid cells following T cell activation (22, 23), the development of NO-producing cells was assessed in the
spleens of Cy-treated mice. As shown in Fig. 2A, large amounts of nitrite,
an end product of NO, were present in spleen cell cultures derived from
mice treated with Cy. This production was achieved between 7–14 days
post-treatment in cultures stimulated with IFN- plus 1C10, a CD40
agonist mAb (30). During this time a strong increase in
WGA+ cells was seen, and nitrite production was
closely associated with their presence in the very same spleens (Fig. 2B). Spleen cells from control mice (Cy-untreated) were
<20% of WGA+ cells and were unable to produce
nitrite above the detection limits of the assay (3 µM; data not
shown). On day 7 after Cy treatment, cells of myeloid lineage were
expanded in the spleen, as detected by GR-1 (Ly-6G), CD11b (Mac-1),
ER-MP54, ER-MP12, and ER-MP20 markers (Fig. 3). Virtually all NO-producing cells in
the spleens of Cy-treated mice that were responding to both IFN-
plus 1C10 were plastic nonadherent, of low density, and recovered in
the Fr3 fraction of Percoll gradients (data not shown). As shown in
Fig. 4, almost all (>90%) Fr3 spleen
cells from Cy-treated mice were both ER-MP12 and ER-MP20 positive.
Taken together these results were consistent with the development of
early myeloid cells in Cy-treated mice, which were producing NO upon
activation by T cell-derived signals.
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Production of NO by bone marrow-derived natural suppressor cells
is strongly antiproliferative for lymphocytes (22). As
shown in Table I, this was also
the case when testing ET cell proliferation in cocultures with Fr3
cells derived from Cy-treated mice. A strong suppression of thymidine
uptake was observed in cultures stimulated with exogenous IFN- plus
1C10, in which nitrite was accumulated in supernatant. Addition of
LMMA, an inhibitor of NO synthesis, to these cultures prevented both
growth inhibition and nitrite production, suggesting the involvement of
NO in ET cell growth suppression. The sensitivity of these cells to NO
was confirmed by adding an NO donor agent (SNAP) to ET cell cultures.
As shown in Fig. 5, ET cell proliferation
was strongly inhibited when achieving comparable amounts of nitrite as
those obtained by Fr3 cells activated with IFN- plus 1C10.
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To test the involvement of NO in tumor suppression achieved in
mice treated with Cy plus immune cell transfer, these animals were
treated with LMMA at the site of tumor inoculation. As shown in a
representative experiment (Fig. 6), ET
growth increased significantly in LMMA-treated mice despite the
combined therapy. By contrast, no enhancement of tumorigenicity could
be seen in control mice (Fig. 6), pointing to NO being involved in the
mechanism of host resistance following Cy treatment plus immune cell
transfer.
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To assess whether early myeloid cells could be present at the
tumor site in Cy-treated mice, tissue sections obtained 2 or 6 days
after challenge with ET cells were analyzed histologically. Fig. 7 shows the results of a representative
experiment. As can be seen, a significant increase in cells stained
with ER-MP54 mAb was scored in mice treated with Cy plus immune cell
transfer. This was the case whether cell transfer was conducted
systemically (Fig. 8A) or
locally (Fig. 8B). It is noteworthy that this increase was
lacking when mice were treated with Cy plus control lymphocytes as well
as when cells were treated with Cy or lymphocytes from immune mice
alone (Fig. 8). As shown in the same figure, the number of neutrophils,
mainly immature forms, increased in most sections from Cy-treated mice
regardless of transfer with immune spleen cells. To assess the presence
of NO-producing cells at the site of the tumor challenge, sections were
also stained for cytoplasmatic iNOS. As shown in Fig. 9, iNOS-positive cells were abundant in
tissue sections from mice treated with the combined therapy in a
Winn’s assay, but not when treated with Cy or immune spleen cells
alone (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study shows, in line with previous descriptions, that a synergism between Cy and spleen cells from mice immunized with tumor cells does exist, resulting in a strong inhibition of tumor growth. It is of note that in the current study Cy was administered 1 wk before tumor challenge, i.e., into tumor-free mice; therefore, its effect on tumor-induced suppressor cells can be ruled out. This agrees with the data reported by Proietti et al. (7), which do not support that the action of Cy is a mere inhibitor of putative suppressor T cells. Instead, these authors suggested that Cy could act by promoting the outgrowth and survival of the transferred lymphocytes by virtue of the cytokine rebound events induced in Cy-treated animals (7, 8). However, this fact alone also seems unlikely in our experimental system, because the effect of the combined therapy was seen when immune cells were coinoculated with tumor cells (Winn’s assay). With this approach the inhibition of tumor growth associated with the combined therapy was evidenced as a very early event, detectable microscopically on the first days after tumor inoculation.
Our results show an increase of ER-MP54+ cells
infiltrating the site of tumor challenge at very early stages of tumor
development. This cytoplasmatic marker is specific for immature myeloid
cells (29) and, therefore, demonstrates the presence of
these in the site of potential tumor development. Importantly, such an
increase was only seen in mice subjected to the combined therapy with
immune, but not control, spleen cells, whether administered
systemically or locally. Moreover, no increase was seen when mice were
treated with immune cell transfer or Cy alone. Nevertheless, Cy alone
did induce a rise in early myeloid cells (WGA+,
Gr-1+, Mac-1+, ER-MP12+,
ER-MP20+, ER-MP54+) in the
spleen 1–2 wk after treatment. This indicates that the transferred
spleen cells from tumor-immunized mice are required for the recruitment
of Cy-induced early myeloid cells at the tumor site. Lymphocytes
specific for ET cells are obviously present at the site of tumor
inoculation in Winn’s assays and probably following their systemic
transfer as described in other tumor models (4, 36). In
this regard most studies emphasize that CD4 T cells are the immune
cells responsible for the antitumor effect associated with the combined
therapy with Cy (5, 6, 7, 8, 9). The fact that these lymphocytes
are a rich source of chemokines with activity on monocytes and murine
granulocyte-macrophage progenitors (37), such as
macrophage inflammatory protein-1
or -1
(38), might
explain that ER-MP54+ cell influx.
A strong inhibition of tumor growth was observed with the combined
therapy. Although the present results do not make clear the actual
involvement of Cy-induced early myeloid cells in tumor suppression,
some findings point in this direction. First, the results show that
early myeloid cells developed after Cy treatment may strongly inhibit
ET cell growth in vitro. This inhibition was mediated by NO released by
ER-MP12+ ER-MP20+
spleen cells (Fr3 cells) upon their activation with IFN- plus CD40
ligation, i.e., T cell-derived signals. This fits with the ability of
bone marrow-derived early myeloid cells (ER-MP12+
ER-MP20+) to release large amounts of NO
following this activation (23) and with a recent study
that reports similar findings when assessing the spleens of mice
treated with Cy (24). The fact that LMMA administered at
the site of tumor inoculation results in a loss of tumor resistance
supports the involvement of NO in the tumor inhibition mechanism. In
this sense, iNOS-positive cells were found only in tissue sections
containing high numbers of ER-MP54+ cells, i.e.,
in mice subjected to the combined therapy. It should be noted that ET
cells, although sensitive to NO as we show here, are highly resistant
to NK activity (32) and probably to CD8-mediated
cytotoxicity because of their lack of H-2 Ags (39), a
feature often described in tumor cells (40). Moreover,
spleen cells from ET-immunized mice were completely unable to inhibit
ET cell growth in cocultures performed in vitro (data not shown).
Therefore, the current results suggest that Cy, which mobilizes and
expands hemopoietic progenitors from bone marrow (41, 42),
induces early myeloid effector cells that may inhibit tumor cell growth
by releasing NO upon activation. The transferred immune spleen cells
might have a double role in this task, as they could be required not
only for effector cell recruitment but also for effector cell
activation at the tumor site. This latter is consistent with the fact
that early myeloid cells are CD40+ and produce NO
following T cell signals (23, 24). Neutrophils are also
present in Cy-treated mice with a progressive growing tumor; therefore,
their active role in tumor suppression seems to be limited.
Nevertheless, they might act in conjunction with NO-producing cells in
those mice subjected to the combined therapy, for example by producing
superoxide anion, which in the presence of NO forms peroxynitrite, a
highly reactive oxidant with strong cytotoxic properties
(43). In fact, such a mechanism has been described
recently to explain some pathogenic effects of Cy following allogeneic
bone marrow transplants (27), and NO has been involved in
the pathogenesis of Cy-induced hemorrhagic cystitis
(26).
The above results may suggest that inhibition of tumor cell growth
should be enhanced in other conditions where outgrowth of immature
myeloid cells is seen, e.g., in those described for natural suppressor
cell induction, provided that T cell signals are given. In this regard,
natural suppressor cells are also induced following irradiation
(14, 15), which, not surprisingly, is an alternative
conditioning regimen for adoptive immunotherapy (44, 45).
It is noteworthy that radiation-induced enhancement of antitumor
immunity has been suggested, in common with Cy, to be dependent on
suppressor cell removal (44), a fact not supported by
other studies (45). Graft-vs-host disease is another
condition where natural suppressor cells are developed
(14). This condition is associated with the
graft-vs-leukemia effect, because it is greatly impaired in their
absence (46). Moreover, IL-2 enhances the
anti-leukemia effect of T cell-depleted allografts
(46). Because this cytokine expands myeloid progenitors
(47), a cell population with IL-2R (48), and
activates bone marrow-derived natural suppressor cells (21, 49), the antitumor effect of these cells should also be
considered. Reports associating the antitumor response of IL-2 therapy
with NO production (50, 51) or describing an antitumor
synergism between Cy and IL-2 (52) are consistent with
this hypothesis. Finally, another condition in which natural suppressor
cells are expanded is in the late stages of tumor development
(16, 53, 54, 55), probably due to stimulation of myelopoiesis
in tumor bearers (56, 57). Thus, tumor-induced natural
suppressor cells might potentially inhibit tumor cell growth, as
described for those suppressor cells found in normal bone marrow
(17, 18) or like those shown here. Hence, natural
suppressor cells with features of early myeloid cells are found
infiltrating progressively growing tumors, associated with impaired
antitumor responses (54, 58). However, these cells release
small amounts of NO (54), which may indicate poor cell
activation and/or a down-regulation of NO production, e.g., by
tumor-derived factors such as TGF- (59, 60). Production
of NO by these cells may not be impaired in the very early stages of
tumor development, in which rather low local concentrations of
inhibitors should be expected. However, in these early stages
tumor-induced specific responses and natural suppressor cells have not
yet been developed (16, 53). This may not be the case if a
second inoculation of tumor cells is conducted in animals bearing an
advanced tumor. This latter might account for the still unexplained
phenomenon of concomitant tumor immunity, i.e., the resistance of
secondary tumor challenge vs the progressive growing primary tumor
described in mice bearing large tumor burdens (61, 62).
The facts that concomitant resistance is a very early event after the
second tumor inoculum, that it has been associated with a strong
monocyte/macrophage infiltration (63, 64), and that it is
sensitive to LMMA treatment (B. Peláez, unpublished observations)
appear to support this view.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. José L. Subiza, Department of Immunology, Hospital Clinico San Carlos, 28040 Madrid, Spain. E-mail address: jlsub{at}retemail.es
3 Abbreviations used in this paper: Cy, cyclophosphamide; ET, Ehrlich tumor; Fr, Percoll spleen fraction; LMMA, NG-monomethyl-L-arginine monoacetate salt; SNAP, S-nitroso-N-acetyl-D,L-penicilamine; iNOS, inducible NO synthase; WGA, wheat-germ agglutinin.
Received for publication August 16, 2000. Accepted for publication March 5, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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;
hematoxylin-eosin) and ER-MP54 staining (
; immunoperoxidase)
on adjacent sections. Cells were counted on coded samples using
an image analyzer system (Leica Q 500 IW) on 22,000 µm2.
The results are the mean ± SD of three mice per group.