Regulation of Tumor-induced Myelopoiesis and the Associated Immune SuppressorCells in Mice Bearing Metastatic Lewis Lung Carcinoma by Prostaglandin E21
M. Rita I. Young,2 Melvin E. Young, and Kimberly Kim
Department of Research Services, Edward J. Hiñes,Jr., Veterans Administration Hospital, Hiñes,Illinois 60141 [M. R. I. Y., M. E. Y., K. K.J, and Department ofPathology, Loyola University Stritch School of Medicine, Maywood, Illinois 60153 [M. R. I. Y.J
ABSTRACT
The in vitro and in vivo effects of prostaglandin I^ ( l'( ; I-'.,) and of its
stable analogue, 16,16-dimethyl-PGE2 (dmPGE2), on myelopoiesis andon the myelopoiesis-associated immune suppressor cell activity of micebearing metastatic variant Lewis lung carcinoma (LLC-C3) tumors are
assessed. In vitro studies showed a reduced susceptibility of bone marrowmyeloid progenitor cells (CFC) from LLC-C3 tumor bearers versusnormal mice to the growth-inhibitory effects of PGE2. When added tococultures of bone marrow cells with LLC-C3 supernatant*, PGE2 less
ened the frequency of CEC and slightly reduced the generation of bonemarrow immune suppressor cells. In vivo studies showed that 4 dailyinjections of dmPGE2 into LLC-C3 tumor-bearing mice caused some
reduction in femoral bone marrow CEC and had an insignificant effect onbone marrow suppressor cell activity. In contrast to the relative insensi-
tivity of bone marrow cells of tumor bearers to the effects of PGE2, invitro studies showed that CFC formation by spleen cells of tumor bearerswas readily inhibited by PGE2. Likewise, in vivo studies showed thatspleen cells of dmPGEj-treated LLC-C3-bearing mice had a reduction in
cellularity, CEC, and the level of spontaneous proliferation; a reductionin suppressor cell activity; and an increase in blastogenesis. Thus, short-term dmPGE2 treatment of LLC-C3-bearing mice limited the tumor-
induced splenic myelopoiesis and reduced the associated splenic immunesuppressor cell activity.
INTRODUCTION
The progressive growth of tumors in patients and experimental animals results in an aberration in the regulation of bothmyelopoiesis and immunity. For example, growth in mice of avariety of tumor types, including TS/A mammary adenocarci-noma, LLC,3 or 4T00.1 plasmacytoma, resulted in splenomegaly and an increase in myeloid progenitor cells (CFC) (1-10).This tumor stimulation of myelopoiesis was associated withtumor cell production of CSFs. Tumor stimulation of myelopoiesis in mice bearing BMT-11 fibrosarcomas enhanced theability of injected B16 melanoma tumor cells to colonize andto metastasize (11).
Tumor growth in a host frequently results in reduced immunecompetence and an increased appearance of immune suppres-sive cells (12-17). Increased suppressor T-lymphocyte activitywas apparent in lymph nodes adjoining melanotic tissue and inthe spleens of mice bearing a variety of tumor types (18-20).Suppressive activity of monocytes and macrophages becamemore prominent during progressive growth of X5563 orMOPC-315 plasmacytomas, methylcholanthrene-induced fibrosarcomas, and of LLC tumors (17, 21-24).
Received 6/23/88; revised 8/15/88; accepted 8/18/88.The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This study was supported in part of the Medical Research Services, HiñesV.
A. Hospital, Hiñes,IL, and by the National Cancer Institutes of Health GrantÇA4S080.
2To whom requests for reprints should be addressed, at Pathology Research(151-Z2), HiñesV. A. Hospital, Hiñes,IL 60141.
3The abbreviations used are: LLC, Lewis lung carcinoma; CSF, colony-stimulating factors; PGE2, prostaglandin E2; FBS, fetal bovine serum; dmPGE2,16.16-dimethyl-PGE2; PWMSCS, pokeweed mitogen-stimulated spleen cell supernatant; ConA, concanavalin A.
In our studies, we have shown that the decline in immunecompetence of mice with progressively growing metastaticLLC-C3 tumor cells was associated with the appearance of asequence of immune suppressor cell populations (5, 16, 17, 24,25). These suppressor cells were inhibitory to the activities ofT-lymphocytes and natural killer cells, both of which are knownto be important components of the host anti-tumor defensesystem. First the immune suppression was mediated by PGE2-producing adherent splenic and peritoneal macrophages (16,17, 24). However, as tumors became large (>3 g), the suppres-sive activity of mature adherent macrophages declined as didtheir production of PGE2. The decline of the PGE2-mediatedimmune suppressor mechanism coincided with an increasedstimulation of myelopoiesis and the appearance of a populationof immature bone marrow-derived immune suppressor cells (5,25). The myelopoiesis stimulation resulted in splenomegaly andin increased numbers of progenitor cells in both the bonemarrow and spleen. The myelopoiesis-associated suppressorcells resembled immature cells of the monocyte lineage, andtheir presence was associated with an increased frequency ofmonocytic cells in the spleen and peripheral blood (5).
The regulation of myelopoiesis is normally controlled bypositive mediators such as the M-CSF, GM-CSF, G-CSF, andinterleukin 3 (26-28), and negative regulators such as acidicisoferritins, interferons, and prostaglandins (29-34). Administration of PGE2 to mice or the addition of macrophage-derivedPGE to in vitro bone marrow cultures resulted in a reductionin myeloid CFC (35, 36). The PGE can also modulate expression of la antigen on stem and myeloid progenitor cells and, inturn, regulate the sensitivity of these cells to inhibition by acidicisoferritins (37-39).
In the current study, a known negative regulator of myelopoiesis, PGE2, was used to minimize the myelopoiesis stimulation which was prominent in mice bearing LLC-C3 tumors.By doing so, we were able to minimize the myelopoiesis-associated immune suppressor activity of spleen cells and, consequently, augment the T-lymphocyte immune competence of theLLC-C3 tumor bearers.
MATERIALS AND METHODS
Medium. The medium used was RPMI-1640 containing 100 units/ml of penicillin, 100 ^g/ml of 4-(2-hydroxyethyl)-l-piperazineethane-suifonic acid buffer solution, 5 x 10~5M 2-mercaptoethanol, 2 HIML-
glutamine, and 10% endotoxin-free FBS (Hyclone Laboratories, Logan,UT).
Mice. Six- to 8-wk-old male C57BL/6 mice were used for all studies.The mice were obtained from Cumberland View Farms (Clinton, TN)and then housed at the HiñesV. A. animal research facility.
LLC-C3 Tumor Cells (40). Cloned metastatic variant cells of theLewis lung carcinoma, LLC-C3, were used in all studies. These cellsdo not produce immune suppressive factors and produce only minimallevels of PGE2. However, the LLC-C3 cells secreted CSF activitieswhich stimulate proliferation of monocytic and monocytic-granulocyticbone marrow CFC (5). In the present studies, the LLC-C3 tumor cellswere implanted into mice by dorsal s.c. injection of 5 x IO5cells.
Culture of Bone Marrow Cells with LLC-C3 Supernatant. Normal
bone marrow cells were suspended to 5 x 106/ml in medium only ormedium containing 20% supernatant from 24-h cultures of 1 x 106/mlof LLC-C3 cells. In some of these bone marrow cultures, PGE2 was
added to yield 1 ng/ml or 0.1 ng/ml. After 3 days of culture, both theadherent and nonadherent cells were collected and used in CFC andsuppressor cell assays.
Treatment of Mice with dmPGE2. At 3 wk after LLC-C3 tumorimplantation, when tumors were approximately 20 mm in diameter,normal and tumor-bearing mice were treated daily for 4 days by i.p.injection of diluent or 10 /¿gof dmPGE2 (Upjohn Diagnostics, Kala-mazoo, MI). On the day following the last dmPGE2 treatment, micewere sacrificed, and their spleen cells and femoral bone marrow cellswere used. Studies were conducted with at least 5 mice/group.
CFC Assay (41). Bone marrow cells (7.5 x 10") or spleen cells (7.5x 10*) from individual mice were placed into each 35 x 10-mm tissueculture dish in 1 ml of semisolid supplemented RPMI-1640 medium
containing 20% FBS and 0.3% agar (Bacto Agar; Difco Laboratories,Detroit, MI). PWMSCS was used as a source of CSF (42) at a finalconcentration of 10%. This preparation of murine CSF, which was freeof known inhibitory molecules, stimulated colonies containing granu-
locytes (approximately 10%), macrophages (approximately 30%), andgranulocytes plus macrophages (approximately 50%). In some assays,various concentrations (100 pg/ml to 1 Mg/ml) of PGE2 were added tothe dishes. The colonies (>50 cells) were counted after 6 days of culture.
T-Lymphocyte Blastogenesis. The T-lymphocyte activity was assessedby culturing 2 x 10s spleen cells for 3 days in microtiter plates withCon-A. For the last 18 h of culture. 1 ¿¿Ciof [3H]thymidine was addedto each well. The amount of [3H]thymidine incorporated by the spleen
cells was measured in a liquid scintillation counter. All assays wereconducted in triplicate on mice from groups of at least 5 mice each.Each assay was repeated at least 3 times.
Suppressor Cell Assay. The presence of immune suppressor cells wasmeasured by the capacity to suppress the T-lymphocyte blastogenicresponse of normal spleen cells to Con-A. Prior to being added toresponder spleen cells, the suppressor cells were irradiated, washed,and added at different ratios to 2 x IO5normal responder spleen cellsand 4 fig/ml of Con-A.
Analysis of Data. The Student t test was used to determine thesignificance of the differences between values.
RESULTS
In Vitro Effect of PGE2 Formation by Bone Marrow andSpleen Cells of LLC-C3 Tumor-bearing Mice. The frequency ofCFC in the bone marrow and spleen cells of mice bearing largemetastatic LLC-C3 tumors was increased to 1.6-fold and 5.5-fold, respectively, that of normals (Table 1). This increase in
the frequency of CFC contributed to an increase in the numberof CFC per femur or per spleen. For example, the number ofCFC per femur of tumor bearers was increased to 40.2 ±1.9 xIO3as compared to 15.6 ±2.0 x IO3 for normals. The number
of CFC per spleen of tumor bearers was increased to 25.3 ±4.4 x IO3 as compared to 2.2 ±1.5 x IO3 for normals. The
addition of PGE2 to the cultures of bone marrow or spleen cellsresulted in a dose-dependent reduction in the CFC formation.This was evident for cells obtained from either normal mice orLLC-C3 bearers. There was, however, a reduced susceptibilityof the bone marrow cells from tumor bearers versus normals tothe inhibitory effects of PGE2. A 50% inhibition of CFC formation by bone marrow cells of normal mice was achieved withless than 1 ng/ml of PGE2, while the 50% inhibitory PGE2dose was approximately 460 ng/ml for bone marrow cells ofLLC-C3-bearing mice. In contrast to the relative insensitivityof bone marrow cells of tumor bearers to the inhibitory effectsof PGE2, CFC formation by spleen cells of tumor bearers wasmore readily inhibited by PGE2 with the 50% inhibitory PGE2dose being approximately 22 ng/ml.
In Vitro Effects of PGE2 on the Frequency of Progenitor Cellsand on the Generation of Suppressor Activity in Cultures of BoneMarrow Cells with LLC-C3 Supernatant. Our previous studiesshowed that LLC-C3 tumor cells secrete CSFs and that theLLC-C3 supernatants induced the appearance of suppressorcells from normal bone marrow cells (5). Culturing bone marrow cells for 3 days in medium containing 20% LLC-C3 supernatant resulted in the persistence of bone marrow progenitorcells which subsequently were able to grow into colonies in thesoft agar CFC assay (Table 2), and in the generation of immunesuppressor cells which were inhibitory to T-lymphocyte activation (Table 3). The immune suppressor cells which were inducedby culture with LLC-C3 supernatants mediate their suppressiveactivities through a prostaglandin-independent mechanismwhich was not influenced by the addition of the prostaglandinsynthesis inhibitor, indomethacin. For example, in one studythe tumor supernatant-cultured bone marrow cells inhibitednormal spleen cell blastogenesis to Con-A by 82% in the absenceof indomethacin and by 78% in the presence of indomethacin.In contrast to the effects of culturing bone marrow cells withtumor supernatant, culturing bone marrow cells with only medium resulted in a loss of progenitor cells and in marginal levelsof suppressor cell activity. The addition of PGE2 to bonemarrow cells cultured with either LLC-C3 supernatant or with
Table 1 In vitro effect ofPGE2 on colony formation by bone marrow and spleen cells from LLC-C3-bearing miceInto each 35-mm dish were added various amounts of PGE2 and either 7.5 x IO4 bone marrow cells or 7.5 x 10* spleen cells from normal or LLC-C3 tumor-
bearing mice with 10% (v/v) PWMSCS as a source of CSF. The percentage of the number of colonies which formed in the absence of added PGE2 and the significanceof this difference are also shown.
Table 2 In vitro reduction by PGE2 of the number of progenitor cells in culturesof bone marrow cells with LLC-C3 supernatant
Normal bone marrow cells were cultured for 3 days with medium or with 20%LLC-C3 supernatant in medium with either no added PGE2 or with O.I ng/ml or1.0 ng/ml of PGE2. These cells were then collected and used in a CFC assay byadding 7.5 x IO4cells to 35-mm dishes with 10% (v/v) PWMSCS as a source of
CSF.
Cells in CFCassayFreshBM*BM
precultured withMedium
+ 0.1 ng/ml of PGE2+ 1.0 ng/ml ofPGE2BM
precultured withLLC-C3 supernatant
+ 0.1 ng/ml of PGE2+ 1.0 ng/ml of PGE2CFC/dish83.7
±8.4r18.3
±1.513.3 ±0.69.0 ±2.659.0
±5.244.3 ±4.230.3 ±5.7PGE2
effect,(%)"-27
-51-25
-49
°Values for the number of CFC formed by PGE2-precultured cells which are
significantly different (P < 0.05) from the CFC values for cells precultured in theabsence of added !'(rl are expressed as the percentage of change due to PGE2.
ft BM, bone marrow.' Mean ±SD from at least 3 dishes per group.
medium alone caused a reduction in the frequency of CFC(Table 2). The addition of PGE2 to these bone marrow cellcultures also caused a slight reduction in the generation ofsuppressor cell activity by the LLC-C3 supernatant and in theless prominent suppressor cell activity which was present afterculture with only medium (Table 3).
Bone Marrow and Spleen Cellularity and Number of CFCfollowing Treatment of LLC-C3-bearing Mice with dmPGE2.The effect of treating LLC-C3 tumor-bearing mice with dm-PGE2 on the myelopoiesis parameters of the femoral bonemarrow and spleens was evaluated (Table 4). The frequency ofbone marrow CFC and the number of CFC/femur in LLC-C3bearers were increased to 1.9-fold and 1.7-fold, respectively,that of normal mice. Treatment of LLC-C3-bearing mice withdmPGEi decreased the frequency of bone marrow CFC by 32%and the number of CFC per femur by 38%. Similarly, treatmentof normal mice with dmPGE2 decreased the frequency of bonemarrow CFC by 26% and the number of CFC per femur by24%. The reduction in CFC/femur in dmPGE2-treated tumor-bearing and normal mice was due to the reduction in the
frequency of bone marrow CFC and not due to a reduction infemoral cellularity which was not significantly altered by eitherthe presence of the tumor or by the treatment with dmPGE2.
In contrast to the bone marrow, both the spleen cellularityand the frequency of CFC of LLC-C3-bearing mice were increased resulting in over a 20-fold increase in the number ofCFC per spleen. Treatment of the LLC-C3 bearers with dm-PGE2 resulted in a 56% decrease in CFC per spleen which wasdue to both a decrease in the frequency of CFC and in thenumber of nucleated cells in the spleen. Treatment of normalmice with dmPGE2 also decreased the number of cells perspleen but caused a slight increase in the frequency of splenicCFC. Consequently, dmPGE2 treatment of normal mice hadno effect on the number of CFC per spleen.
In a separate study, the effect of dmPGE2 treatment on thespleen cell incorporation of [3H]thymidine during the 18 hfollowing removal from tumor-bearing or normal mice wasmeasured and was shown to agree with the above results of theCFC assay. In the absence of any stimulants, the [3H]thymidineincorporated (cpm) by spleen cells of placebo-treated normaland LLC-C3-bearing mice was 13,189 ±2,551 and 120,100 ±5,482, respectively. The [3H]thymidine incorporated by spleencells of dmPGE2-treated normal and LLC-C3-bearing mice wasreduced, respectively, to 9,571 ±2,719 (not significantly different) and to 81,457 ±11,622 (P < 0.01 ).
Effect of Treating Normal and LLC-C3-bearing Mice withdmPGE2 on Their Spleen Cell Blastogenic Response. The effectof treating normal and tumor-bearing mice with dmPGE2 ontheir spleen cell blastogenic response to various doses of Con-A was measured (Table 5). Treatment of normal mice withdmPGE2 had no effect on blastogenesis in response to 4 /ug/mlof Con-A and inhibited blastogenesis in response to 2 or 1 ¿tg/ml of Con-A by 24% and 26%, respectively (P< 0.05). Placebo-treated LLC-C3 bearers were minimally able to respond toCon-A with their blastogenesis being reduced by over 60% ascompared to that of normal mice. In contrast to the immunesuppressive effect of dmPGE2 treatment in normal mice, treatment of tumor-bearing mice with dmPGE2 resulted in a significant restoration of their Con-A responsiveness. For example,dmPGE2 treatment of tumor bearers restored their responsiveness to 4 jig/ml of Con-A from 37% to 75% of the response bynormals.
Table 3 In vitro inhibition of the generation of bone marrow suppressor cells by culture with PGEjNormal bone marrow cells were cultured for 3 days with medium or with 20% LLC-C3 supernatant in medium with either no added PGE2 or with O.I ng/ml or
1.0 ng/ml of n .1 The ability of these cells to suppress normal spleen cell blastogenesis to Con-A was measured and compared to that for freshly isolated bonemarrow cells.
" BM, bone marrow." Ratio of responder normal spleen cells to added suppressor cells.' Mean ±SD of at least triplicate cultures (cpm).11Numbers in parentheses, significant (P < 0.05) levels of suppressor cell activity expressed as the percentage of suppression of the normal spleen cell response.' Significant (/' < 0.05) reduction in suppressor activity of PGEi-cultured cells as compared to that of cells cultured in the absence of added I'<. I
Table 4 Effect afin vivo administration ofdmPGEi to LLC-Ci-bearing mice on the myelopoietic parameters of their femoral bone marron1 and spleensBone marrow cells (7.5 x IO4)or spleen cells (7.5 x 10') from individual mice were seeded in triplicale into 35-mm dishes with 10% (v/v) PWMSCS. After 6 days,
the number of CFC per dish was determined. In Experiments 3 to 5, the numbers of nucleated cells in the femoral bone marrow or in the spleen of individual micewere also determined and the numbers of CFC per organ were then calculated.
°Mean ±SEM.* Values for dmPGE2-treated mice is significantly different (P < 0.05) from the value for placebo-treated mice.' Numbers in parentheses, values which are significantly different (P < 0.05) from those for normal placebo-treated mice expressed as the percentage of the normal
value.
Table 5 Effect ofdmPGEi treatment ofLLC-Ci tumor bearers on their Con-A blastogenic response
Con-A Spleen cell(¡.•j.milsource4
NormalsLLC-C3bearers2
NormalsLLC-C3bearers1
NormalsLLC-C3 bearersCon-A
response of mice treatedwith"Placebo183,
160 ±6,424''
68,368 ±12,906(37/227,330
±36,47146,459 ±1.354(20)196,463
±6,95818,584 ±2,936 (9)dmPGEj1
77,387 ±10,811136,730 +5,096(75)173,037+
10,315(76)136,373 +9,682(60)1
44,623 ±11,537(74)77,162 ±8,364 (39)%of
change*-3+99-24
+194-26
+315P
value'NS'
<0.01<0.05
<0.001<0.01
<0.001°The Con-A blastogenic response of individual control and LLC-C3 tumor-bearing mice which had been treated with placebo or with dmPGEj was measured.
Each treatment group contained 8 mice.* The percentage of change in Con-A blastogenesis due to dmPGE; treatment of mice.' Level of significance in the difference between the response of dmPGE2-treated mice as compared to the response of placebo-treated mice.d Mean ±SEM (cpm).' NS, not significant.^Numbers in parentheses, values which are significantly different (P < 0.05) from those for normal placebo-treated mice expressed as the percentage of the normal
value.
Table 6 Effect ofdmPGE2 treatment ofLLC-Ci tumor bearers on their suppressor cell activityCon-A blastogenic response by spleen cells"
" The effect of bone marrow and spleen cells from placebo- or dmPGEj-treated normal or LLC-C3-bearing mice on the Con-A responses of normal spleen cells
was assessed. Each treatment group contained 8 mice.* Ratio of responder normal spleen cells to added suppressor cells.' N/A, not available; BM, bone marrow.**Mean ±SEM of 3 experiments.' Significant (P < 0.05) levels of suppressor cell activity expressed as the percentage of suppression of the normal spleen cell response by the added cells.* Value for dmPGE2-treated mice is significantly (P < 0.05) different from the value for placebo-treated mice.
Effect of dmPGEz Treatment of Normal and LLC-C3-bearingMice on the Suppressor Activity of Their Bone Marrow andSpleen Cells. Studies were conducted to determine if dmPGEjtreatment of normal and LLC-C3-bearing mice would minimizethe levels of immune suppressor activity of their bone marrowand spleen cells (Table 6). While bone marrow cells of normalmice had some suppressor cell activity, causing 32% suppression of normal spleen cell blastogenesis, bone marrow cells ofLLC-C3-bearing mice had more suppressor activity and suppressed blastogenesis by 50%. Treatment of normal mice with
dmPGEi caused a complete elimination of suppressor activityof their bone marrow cells. In contrast, dmPGE2 treatment oftumor-bearing mice tended to only minimally lessen their bonemarrow suppressor activity (not statistically significant).
The effects of dmPGE2 administration to tumor bearers onthe suppressor activity of their spleen cells differed from theresults described above for bone marrow cells. No suppressiveactivity was present in spleen cells prepared from either placebo-treated or dmPGE2-treated normal mice. In contrast, spleencells of LLC-C3-bearing mice suppressed normal spleen cell
blastogenesis by 41%. This suppressive activity was abolishedby treatment of LLC-C3-bearing mice with dmPGE2. Similarresults were observed at both of the ratios at which suppressorcells were added to normal splenic responder cells.
DISCUSSION
Progressive growth of tumors results in the appearance ofimmune suppressor cells which limit the antitumor effectormechanisms of the host (12, 16-21). Our studies have shownthat growth of LLC-C3 tumors profoundly suppresses hostimmune competence. This immunosuppression is mediated byhost suppressor mechanisms, since the tumor itself does notproduce immune suppressive factors such as PGE2. However,the LLC-C3 tumor cells do secrete CSFs. Our studies (5, 17,24, 25) have suggested the following cascade of tumor-inducedhost suppressor mechanisms which overlap with mechanismsthat regulate myelopoiesis. During the initial and intermediateperiods of LLC-C3 growth, mature adherent macrophages mediate immune suppression by producing increased amounts ofPGE2. So long as the macrophages produce elevated levels ofPGE2, they also block myelopoietic stimulation by the tumor-derived CSF. During the later phases of tumor growth, themacrophages lose their suppressive activities and produce onlynominal amounts of PGE2. When macrophage PGE2 production declines, the myelopoietic stimulation by the tumor-derivedCSF becomes evident. Consequently, the myelopoiesis-associ-ated immune suppressor cells also became apparent. As PGE2is a potent negative regulator of myelopoiesis (31-39), it wasused in vitro and in vivo in the presently described studies torestrict the appearance of tumor-induced myelopoiesis and theassociated bone marrow-derived suppressor cells.
The results of in vitro studies showed that PGE2 could beeffective at inhibiting the tumor-induced myelopoiesis in orderto inhibit the appearance of the myelopoiesis-associated immune suppressor cells. The doses at which these effects couldbe observed, 1 ng/ml and 100 pg/ml, are physiologically pertinent, as the plasma PGE2 levels of normal mice and of micebearing large tumors are typically between 530 and 600 pg/ml(Ref. 17 and data not shown). These in vitro studies supportedthe use of PGE2 in LLC-C3 tumor bearers at a time whentumor stimulation of myelopoiesis and the associated appearance of bone marrow-derived suppressor cells were prominent.In the in vivo studies, treatment of tumor bearers with dmPGE2was restricted to 4 daily injections to (a) minimize any directimmune suppressive effects of dmPGE2, and to (b) avoid anychanges in tumor size that would interfere in the assessment ofPGE2 effects on myelopoiesis and suppressor cells. Short-termtreatment of tumor bearers with dmPGE2 resulted in somereduction in femoral CFC and had an insignificant effect onthe presence of immune suppressor bone marrow cells. However, the dmPGE2 treatment of tumor bearers caused a markedreduction in splenic myelopoiesis and a reduction in splenicimmune suppressor cells. The capacity of the dmPGE2 treatments in diminish splenic myelopoiesis and suppressor cellactivity prompts further studies to determine whether theseeffects would be expressed as a measurable reduction in tumorgrowth. These in vivo results were consistent with the results ofin vitro studies showing that bone marrow cells of tumor bearerswere less sensitive to the myelosuppressive effects of PGE2 thanwere either bone marrow cells from normal mice or spleen cellsfrom tumor-bearing mice. It would be of interest to quantitatePGE2 receptors on progenitor cells of tumor bearers versus ofcontrol mice or to compare the levels of responsiveness of
tumor-bearer progenitor cells to PGE2 by assessing either theirintracellular levels of cyclic cAMP or the modulation of theirexpression of la-associated antigen.
The conclusion of our present studies is that short-termtreatment of tumor bearers with PGE2 is efficacious in thespleen in limiting tumor-induced myelopoiesis stimulation andthe presence of myelopoiesis-associated immune suppressorcells.
ACKNOWLEDGMENTS
We thank Dr. Hal E. Broxmeyer of the Indiana University Schoolof Medicine and Dr. Carleton C. Stewart of the Roswell Park MemorialInstitute for their encouragement and advice in the preparation of thismanuscript.
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25. Young, M. R., Young, M. E., and Wepsic, H. T. The effect of recombinantmurine interferon-gamma on the hematopoietic and immunological parameters of mice bearing metastatic Lewis lung carcinoma tumors. Exp. Hematol.(Copenh.), 16: 295-301, 1988.
26. Metcalf, D., Begley, C. G., Williamson, D. J., Nice, E. C, De Lamarter, J.,Mermod, J.-J., Thatcher, D., and Schmidt, A. Hemopoietic responses inmice injected with purified recombinant murine GM-CSF. Exp. Hematol.(Copenh.), /5: 1-9, 1987.
27. Bender, J. G., Van Epps, D. E., and Stewart, C. C. A model for the regulationof myelopoiesis by specific factors. J. Leukocyte Biol., 39: 101-111, 1986.
28. Kindler, V., Thorens, B., De Kossodo, S., Allet, B., Eliason, J. F., Thatcher,D., Farber, N., and Vassalli, P. Stimulation of hematopoiesis in vivo byrecombinant bacterial murine interleukin 3. Proc. Nati. Acad. Sci. USA, S3:1001-1005, 1986.
29. Broxmeyer, H. E., Williams, D. E., Lu, L., Cooper, S., Anderson, S. L.,Beyer, G. S., Hoffman, R., and Rubin, B. Y. Suppressive influences of humantumor necrosis factors on bone marrow hematopoietic progenitor cells fromnormal donors and patients with leukemia: synergism of tumor necrosisfactor and interferon-gamma. J. Immunol.. 136:4487-4495, 1986.
30. Broxmeyer, H. E., Juliano, L., Waheed, A., and Shadduck, R. K. Releasefrom mouse macrophages of acidic isoferritins that suppress hematopoieticprogenitor cells is induced by purified L cell colony stimulating factor andsuppressed by human lactofemn. J. Immunol., 135: 3224-3231, 1985.
31. Kurland, J., and Moore, M. A. S. Modulation of hemopoiesis by prostaglan-dins. Exp. Hematol. (Copenh.), 5: 357-373, 1977.
32. Pelus, L. M., Ottmann, O. G., and Nocka, K. H. Synergistic inhibition of
human marrow granulocyte-macrophage progenitor cells by prostaglandin Eand recombinant interferon-alpha, -beta, and -gamma and an effect mediatedby tumor necrosis factor. J. Immunol., 140: 479-484. 1988.
33. Boorman. G. A., Luster, M. I., Dean, J. H., and Luebke, R. W. Effect ofindomethacin on the bone marrow and immune system of the mouse. J. Clin.Lab. Immunol., 7: 117-126, 1982.
34. Nikcevich. D. A., Young, M. R.. Ellis. N. K., Newby, M., and Wepsic, H. T.Stimulation of hematopoiesis in untreated and cyclophosphamide treatedmice by the inhibition of prostaglandin synthesis. J. Immunopharmacol., S:289-313, 1986.
35. Gentile. P. S., and Pelus, L. M. In vivo modulation of myelopoiesis byprostaglandin E2. II. Inhibition of granulocyte-monocyte progenitor cell(CFU-GM) cycle rate. Exp. Hematol. (Copenh.), 15: 119-126, 1984.
36. Kurland, J. I.. Bockman, R. S., Broxmeyer, H. E., and Moore, M. A. S.Limitation of excessive myelopoiesis by the intrinsic modulation of macro-phage-derived prostaglandin E. Science (Wash. DC), 199: 552-555, 1978.
37. Pelus, L. M., Gold, E., Saletan, S.. and Coleman, M. Restoration of responsiveness of chronic myeloid leukemia granulocyte-macrophage colony-forming cells to growth regulation in vitro following preincubation with prostaglandin E. Blood, 62: 158-165, 1983.
38. Lu, L., Pelus, L. M., and Broxmeyer, H. E. Modulation of the expression ofHLA-DR (la) antigens and the proliferation of human erythroid (BFU-E)and multipotential (CFU-GEMM) progenitor cells by prostaglandin E. Exp.Hematol. (Copenh.), 12: 741-748, 1984.
38. Pelus, L. M., Saletan, S., Silver, R. T., and Moore, M. A. S. Expression ofla-antigens on normal and chronic myeloid leukemic human granulocyte-macrophage colony-forming cells (CFU-GM) is associated with the regulation of cell proliferation by prostaglandin E. Blood, 59: 284-292, 1982.
40. Young, M. R., Meunier, J., and Newby, M. Relationships between morphology, dissemination, migration, and prostaglandin E2 secretion by clonedvariants of Lewis lung carcinoma. Cancer Res., 45: 3918-3923, 1985.
41. Broxmeyer, H. E., Cooper, S., Rubin, B. Y., and Taylor, M. W. Thesynergistic influence of human interferon-gamma and interferon-alpha onsuppression of hematopoietic progenitor cells is additive with the enhancedsensitivity of these cells to inhibition by interferons at low oxygen tension invitro. J. Immunol., 135: 2502-2506, 1985.
42. Metcalf, D., Johnson, G. R., and Mandel, F. E. Colony formation in agar bymultipotential hemopoietic cells. J. Cell. Physiol., 98:401-410. 1979.
1988;48:6826-6831. Cancer Res M. Rita I. Young, Melvin E. Young and Kimberly Kim 2Lung Carcinoma by Prostaglandin EImmune Suppressor Cells in Mice Bearing Metastatic Lewis Regulation of Tumor-induced Myelopoiesis and the Associated
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