Convergence of αvβ3Integrin–And Macrophage Colony Stimulating Factor–Mediated Signals on Phospholipase Cγ in Prefusion Osteoclasts

The macrophage colony stimulating factor (M-CSF) and αvβ3 integrins play critical roles in osteoclast function. This study examines M-CSF– and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130Cas, paxillin, and PLC-γ. However, in response to M-CSF, Src−/− pOCs spread and migrate on Vn in an αvβ3-dependent manner. Involvement of PLC-γ activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF–mediated cell spreading. Furthermore, in Src−/− pOCs M-CSF, together with filamentous actin, causes recruitment of β3 integrin and PLC-γ to adhesion contacts and induces stable association of β3 integrin with PLC-γ, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-γ can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-γ is a common downstream mediator for adhesion and growth factor signals. M-CSF–initiated signaling modulates the αvβ3 integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-γ.

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Vav Activation and Function as a Rac Guanine Nucleotide Exchange Factor in Macrophage Colony-Stimulating Factor-Induced Macrophage Chemotaxis

Molecular and Cellular Biology ◽

10.1128/mcb.25.10.4211-4220.2005 ◽

2005 ◽

Vol 25 (10) ◽

pp. 4211-4220 ◽

Cited By ~ 55

Author(s):

Vidya Vedham ◽

Hyewon Phee ◽

K. Mark Coggeshall

Keyword(s):

Sh2 Domain ◽

Small Gtpase ◽

Pleckstrin Homology Domain ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

Wild Type ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Hydrolysis Of ◽

Stimulating Factor

ABSTRACT Signal transduction mediated by phosphatidylinositol 3-kinase (PI 3-kinase) is regulated by hydrolysis of its products, a function performed by the 145-kDa SH2 domain-containing inositol phosphatase (SHIP). Here, we show that bone marrow macrophages of SHIP−/− animals have elevated levels of phosphatidylinositol 3,4,5-trisphosphate [PI (3,4,5)P3] and displayed higher and more prolonged chemotactic responses to macrophage colony-stimulating factor (M-CSF) and elevated levels of F-actin relative to wild-type macrophages. We also found that the small GTPase Rac was constitutively active and its upstream activator Vav was constitutively phosphorylated in SHIP−/− macrophages. Furthermore, we show that Vav in wild-type macrophages is recruited to the membrane in a PI 3-kinase-dependent manner through the Vav pleckstrin homology domain upon M-CSF stimulation. Dominant inhibitory mutants of both Rac and Vav blocked chemotaxis. We conclude that Vav acts as a PI 3-kinase-dependent activator for Rac activation in macrophages stimulated with M-CSF and that SHIP regulates macrophage M-CSF-triggered chemotaxis by hydrolysis of PI (3,4,5)P3.

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Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00309.2007 ◽

2008 ◽

Vol 295 (1) ◽

pp. L114-L122 ◽

Cited By ~ 15

Author(s):

Megan N. Ballinger ◽

Leah L. N. Hubbard ◽

Tracy R. McMillan ◽

Galen B. Toews ◽

Marc Peters-Golden ◽

...

Keyword(s):

Bone Marrow ◽

Host Defense ◽

Receptor Expression ◽

Colony Stimulating Factor ◽

Wild Type ◽

Bacterial Killing ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Impaired host defense post-bone marrow transplant (BMT) is related to overproduction of prostaglandin E2(PGE2) by alveolar macrophages (AMs). We show AMs post-BMT overproduce granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas GM-CSF in lung homogenates is impaired both at baseline and in response to infection post-BMT. Homeostatic regulation of GM-CSF may occur by hematopoietic/structural cell cross talk. To determine whether AM overproduction of GM-CSF influenced immunosuppression post-BMT, we compared mice that received BMT from wild-type donors (control BMT) or mice that received BMT from GM-CSF−/− donors (GM-CSF−/− BMT) with untransplanted mice. GM-CSF−/− BMT mice were less susceptible to pneumonia with Pseudomonas aeruginosa compared with control BMT mice and showed antibacterial responses equal to or better than untransplanted mice. GM-CSF−/− BMT AMs displayed normal phagocytosis and a trend toward enhanced bacterial killing. Surprisingly, AMs from GM-CSF−/− BMT mice overproduced PGE2, but expression of the inhibitory EP2receptor was diminished. As a consequence of decreased EP2receptor expression, we found diminished accumulation of cAMP in response to PGE2stimulation in GM-CSF−/− BMT AMs compared with control BMT AMs. In addition, GM-CSF−/− BMT AMs retained cysteinyl leukotriene production and normal TNF-α response compared with AMs from control BMT mice. GM-CSF−/− BMT neutrophils also showed improved bacterial killing. Although genetic ablation of GM-CSF in hematopoietic cells post-BMT improved host defense, transplantation of wild-type bone marrow into GM-CSF−/− recipients demonstrated that parenchymal cell-derived GM-CSF is necessary for effective innate immune responses post-BMT. These results highlight the complex regulation of GM-CSF and innate immunity post-BMT.

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Growth and differentiation of a human megakaryoblastic cell line, CMK

Blood ◽

10.1182/blood.v74.1.42.42 ◽

1989 ◽

Vol 74 (1) ◽

pp. 42-48 ◽

Cited By ~ 74

Author(s):

N Komatsu ◽

T Suda ◽

M Moroi ◽

N Tokuyama ◽

Y Sakata ◽

...

Keyword(s):

Cell Line ◽

Colony Formation ◽

Culture System ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Hematopoietic Factors ◽

Macrophage Colony ◽

Stimulating Factor

Abstract Recently, a human megakaryoblastic cell line, CMK, was established from the peripheral blood of a megakaryoblastic leukemia patient with Down syndrome. Using this cell line, we studied the proliferation and differentiation of megakaryocytic cells in the presence of highly purified human hematopoietic factors and phorbol 12-myristate-13- acetate (PMA). In a methylcellulose culture system, interleukin-3 (IL- 3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitated colony formation by CMK cells in a dose-dependent manner. The maximum stimulating doses of these factors were 10 and 200 U/mL, respectively. These concentrations were comparable to those that stimulate activity in normal hematopoietic cells. In contrast, granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), and erythropoietin (EPO) had no effects on the colony formation of CMK cells. In a liquid culture system, 20% of the CMK cells expressed glycoprotein IIb/IIIa (GPIIb/IIIa) antigen without hematopoietic factors, whereas 40% of the cells expressed GPIIb/IIIa with the addition of IL-3 and GM-CSF. EPO also slightly enhanced expression of GPIIb/IIIa. On the other hand, PMA inhibited growth of CMK cells and induced most of them to express the GPIIb/IIIa antigen. Furthermore, PMA induced CMK cells to produce growth activity toward new inocula of CMK cells. This growth factor (GF) contained colony-stimulating activity (CSA) in normal bone marrow (BM) cells. The activity was believed to be attributable mainly to GM-CSF, since 64% of this activity was neutralized by anti-GM-CSF antibodies and a transcript of GM-CSF was detected in mRNA from PMA-treated CMK cells by Northern blot analysis. These observations suggest that GM-CSF, as well as IL-3, should play an important role in megakaryocytopoiesis.

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STAT5A-Deficient Mice Demonstrate a Defect in Granulocyte-Macrophage Colony-Stimulating Factor–Induced Proliferation and Gene Expression

Blood ◽

10.1182/blood.v90.5.1768 ◽

1997 ◽

Vol 90 (5) ◽

pp. 1768-1776 ◽

Cited By ~ 131

Author(s):

Gerald M. Feldman ◽

Louis A. Rosenthal ◽

Xiuwen Liu ◽

Mark P. Hayes ◽

Anthony Wynshaw-Boris ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding Proteins ◽

Colony Stimulating Factor ◽

Wild Type ◽

Stat Proteins ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor ◽

In Cells

Abstract Responses of cells to cytokines typically involve the activation of a family of latent DNA binding proteins, referred to as signal transducers and activators of transcription (STAT) proteins, which are critical for the expression of early response genes. Of the seven known STAT proteins, STAT5 (originally called mammary gland factor) has been shown to be activated by several cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5, which are known to play important roles in growth and differentiation of hematopoietic precursors. In this report we have used mice that are deficient in STAT5A (one of two homologues of STAT5) to study the role of STAT5A in GM-CSF stimulation of cells. When bone marrow–derived macrophages were generated by differentiation with macrophage-CSF (M-CSF), exposure of cells from wild-type mice to GM-CSF resulted in a typical pattern of assembly of DNA binding proteins specific for the gamma activation sequence (GAS) element within the β-casein promoter. However, in cells from the STAT5A null mouse one of the shifted bands was absent. Immunoblotting analysis in the null mice showed that lack of STAT5A protein resulted in no alteration in activation of STAT5B by tyrosine phosphorylation. Proliferation experiments revealed that, when exposed to increasing concentrations of GM-CSF, cells derived from the null mice grew considerably more slowly than cells derived from the wild-type mice. Moreover, expression of GM-CSF–dependent genes, CIS and A1, was markedly inhibited in cells derived from null mice as compared with those of wild-type mice. The decreased expression observed with A1, a bcl-2 like gene, may account in part for the suppression of growth in cells from the null mice. These data suggest that the presence of STAT5A during the GM-CSF–induced assembly of STAT5 dimers is critical for the formation of competent transcription factors that are required for both gene expression and cell proliferation.

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Suppressive effect of granulocyte-macrophage colony-stimulating factor on the generation of natural killer cells in vitro

Blood ◽

10.1182/blood.v79.12.3227.3227 ◽

1992 ◽

Vol 79 (12) ◽

pp. 3227-3232 ◽

Cited By ~ 10

Author(s):

K Taguchi ◽

A Shibuya ◽

Y Inazawa ◽

T Abe

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Bone Marrow Cells ◽

Suppressive Effect ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Abstract We investigated the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human granulocyte- CSF (rhG-CSF) on the generation of natural killer (NK) cells in vitro. NK cells were cultured from selected human bone marrow cells obtained after the elimination of mature T and NK cells. rhGM-CSF significantly suppressed the generation of CD56+ cells and NK activity (P less than .01) in a dose-dependent manner. The generation of large granular lymphocytes (LGL) was also suppressed in the presence of rhGM-CSF (P less than .01). In contrast, rhG-CSF had no effect on LGL (P greater than .05). Both rhGM-CSF and rhG-CSF had no influence on the CD56+ cell count in the peripheral blood. These results suggest that rhGM-CSF suppresses the in vitro generation of NK cells.

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Hematopoietic and Lymphopoietic Responses in Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Transgenic Mice Injected With Human GM-CSF

Blood ◽

10.1182/blood.v90.3.1031 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1031-1038 ◽

Cited By ~ 16

Author(s):

I. Nishijima ◽

T. Nakahata ◽

S. Watanabe ◽

K. Tsuji ◽

I. Tanaka ◽

...

Keyword(s):

Transgenic Mice ◽

Blood Cells ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Dose Dependent ◽

Stimulating Factor

Abstract Using a clonal assay of bone marrow (BM) cells from transgenic mice (Tg-mice) expressing the human granulocyte-macrophage colony-stimulating factor receptor (hGM-CSFR), we found in earlier studies that hGM-CSF alone supported the development not only of granulocyte-macrophage colonies, but also of erythrocytes, megakaryocytes, mast cells, blast cells, and mixed hematopoietic colonies. In this report, we evaluated the in vivo effects of hGM-CSF on hematopoietic and lymphopoietic responses in the hGM-CSFR Tg-mice. Administration of this factor to Tg-mice resulted in dose-dependent increases in numbers of reticulocytes and white blood cells (WBCs) in the peripheral blood. Morphological analysis of WBCs showed that the numbers of all types of the cell, including neutrophils, eosinophils, monocytes, and lymphocytes increased; the most remarkable being in lymphocytes that contained a number of large granular lymphocytes (LGLs) in addition to mature T and B cells. However, total cellularity of the BM of the Tg-mice decreased in a dose-dependent manner when hGM-CSF was injected. In sharp contrast to the BM, spleens of the Tg-mice were grossly enlarged. Although all types of blood cells and hematopoietic progenitors increased in the spleen, erythroid cells and their progenitors showed the most significant increase. Increased numbers of megakaryocytes and LGLs were also observed in spleen and liver of the treated Tg-mice. Flow cytometric analysis showed that LGLs expanded in Tg-mice expressed Mac-1+CD3−NK1.1+. The thymus of Tg-mice treated with hGM-CSF exhibited a dose-dependent shrinkage and a remarkable decrease in CD4+CD8+ cells. Thus, hGM-CSF stimulated not only myelopoiesis but also erythropoiesis and megakaryopoiesis of hGM-CSFR Tg-mice in vivo, in accordance with our reported in vitro findings. In addition, hGM-CSF affected the development of lymphoid cells, including natural killer cells of these Tg-mice.

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Erythropoietin receptor haploinsufficiency and in vivo interplay with granulocyte-macrophage colony-stimulating factor and interleukin 3

Blood ◽

10.1182/blood.v99.7.2603 ◽

2002 ◽

Vol 99 (7) ◽

pp. 2603-2605 ◽

Cited By ~ 16

Author(s):

Armin G. Jegalian ◽

Adriana Acurio ◽

Glenn Dranoff ◽

Hong Wu

Keyword(s):

Erythropoietin Receptor ◽

Colony Stimulating Factor ◽

Wild Type ◽

Interleukin 3 ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Definitive Erythropoiesis ◽

Stimulating Factor

Erythropoietin (EPO) and its receptor (EPOR) are critical for definitive erythropoiesis, as mice lacking either gene product die during embryogenesis with severe anemia. Here we demonstrate that mice expressing just one functional allele of the EpoR have lower hematocrits and die more frequently than do wild-type littermates on anemia induction. Furthermore, EpoR+/−erythroid colony-forming unit (CFU-E) progenitors are reduced both in frequency and in responsiveness to EPO stimulation. To evaluate the interaction between EPO and granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3),GM-CSF−/− orIL-3−/− mice were interbred withEpoR+/− mice. Deletion of either GM-CSF or IL-3 also leads to reduction in CFU-E numbers and hematocrits but does not significantly alter steady-state erythroid burst-forming unit numbers. These results suggest EpoR haploinsufficiency and promotion of in vivo erythropoiesis by GM-CSF and IL-3.

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Hematopoietic and Lymphopoietic Responses in Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Transgenic Mice Injected With Human GM-CSF

Blood ◽

10.1182/blood.v90.3.1031.1031_1031_1038 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1031-1038 ◽

Cited By ~ 3

Author(s):

I. Nishijima ◽

T. Nakahata ◽

S. Watanabe ◽

K. Tsuji ◽

I. Tanaka ◽

...

Keyword(s):

Transgenic Mice ◽

Blood Cells ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Dose Dependent ◽

Stimulating Factor

Using a clonal assay of bone marrow (BM) cells from transgenic mice (Tg-mice) expressing the human granulocyte-macrophage colony-stimulating factor receptor (hGM-CSFR), we found in earlier studies that hGM-CSF alone supported the development not only of granulocyte-macrophage colonies, but also of erythrocytes, megakaryocytes, mast cells, blast cells, and mixed hematopoietic colonies. In this report, we evaluated the in vivo effects of hGM-CSF on hematopoietic and lymphopoietic responses in the hGM-CSFR Tg-mice. Administration of this factor to Tg-mice resulted in dose-dependent increases in numbers of reticulocytes and white blood cells (WBCs) in the peripheral blood. Morphological analysis of WBCs showed that the numbers of all types of the cell, including neutrophils, eosinophils, monocytes, and lymphocytes increased; the most remarkable being in lymphocytes that contained a number of large granular lymphocytes (LGLs) in addition to mature T and B cells. However, total cellularity of the BM of the Tg-mice decreased in a dose-dependent manner when hGM-CSF was injected. In sharp contrast to the BM, spleens of the Tg-mice were grossly enlarged. Although all types of blood cells and hematopoietic progenitors increased in the spleen, erythroid cells and their progenitors showed the most significant increase. Increased numbers of megakaryocytes and LGLs were also observed in spleen and liver of the treated Tg-mice. Flow cytometric analysis showed that LGLs expanded in Tg-mice expressed Mac-1+CD3−NK1.1+. The thymus of Tg-mice treated with hGM-CSF exhibited a dose-dependent shrinkage and a remarkable decrease in CD4+CD8+ cells. Thus, hGM-CSF stimulated not only myelopoiesis but also erythropoiesis and megakaryopoiesis of hGM-CSFR Tg-mice in vivo, in accordance with our reported in vitro findings. In addition, hGM-CSF affected the development of lymphoid cells, including natural killer cells of these Tg-mice.

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Macrophage colony-stimulating factor induces substantial osteoclast generation and bone resorption in human bone marrow cultures

Blood ◽

10.1182/blood.v88.7.2531.bloodjournal8872531 ◽

1996 ◽

Vol 88 (7) ◽

pp. 2531-2540 ◽

Cited By ~ 81

Author(s):

U Sarma ◽

AM Flanagan

Keyword(s):

Bone Resorption ◽

Vitamin D3 ◽

Osteoclast Formation ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Human Osteoclasts ◽

Stimulating Factor

Macrophage colony-stimulating factor (M-CSF) is essential for murine osteoclast formation and its role in human hematopoiesis in vitro is not fully defined. Therefore, we have investigated the effect of M-CSF on the formation of human osteoclasts in vitro. M-CSF was found to induce substantial bone resorption and osteoclast formation in a dose-responsive and time-dependent manner above that induced by 1,25 dihydroxyvitamin D3 (1,25 vitamin D3) in cultures of human bone marrow (BM) stromal cells sedimented onto devitalized bone. By day 14 there was a mean of approximately 50% of the surfaces of the bone slices resorbed compared with only 6% in cultures treated with 1,25 vitamin D3 alone. Osteoclasts were identified as 23c6+ cells (an antibody that recognizes the vitronectin receptor), 87.5% of which coexpressed the calcitonin receptor. The number of 23c6+ cells correlated strongly with bone resorption spatially, and in a dose-responsive and time-dependent manner; the correlation coefficient in cultures treated with 1,25 vitamin D3 alone was 0.856 and those treated with both M-CSF and 1,25 vitamin D3 was 0.880. Granulocyte-macrophage colony-stimulating factor, IL-1 beta, IL-3, IL-6, tumor necrosis factor-alpha, transforming growth factor-beta, leukemia inhibitory factor, and IL-11 did not increase bone resorption above that in 1,25 vitamin D3-treated cultures. We also found that 1,25 vitamin D3 increased, to a minor but significant degree, both bone resorption and the concentration of M-CSF in the culture supernatants above that in vehicle-treated cultures, indicating that M-CSF is present in our BM cultures, but that there is insufficient to induce substantial osteoclast formation. These results define a critical role for M-CSF in the formation of human osteoclasts.

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Tumor vaccination with macrophage colony-stimulating factor-producing Lewis lung carcinoma in mice

Blood ◽

10.1182/blood.v88.3.955.bloodjournal883955 ◽

1996 ◽

Vol 88 (3) ◽

pp. 955-961

Author(s):

T Morita ◽

K Ikeda ◽

M Douzono ◽

M Yamada ◽

F Kimura ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Lewis Lung Carcinoma ◽

Lewis Lung ◽

Colony Stimulating Factor ◽

Wild Type ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Expression of various cytokines by cytokine gene-transduced tumor cells has been shown to increase antitumor immunity of tumor-bearing hosts. In the present study, macrophage-colony stimulating factor (M-CSF) cDNA was retrovirally transfected into Lewis lung carcinoma cells (3LL) of C57BL/6 mouse origin, and the effects of M-CSF expression were studied by inoculating syngeneic C57BL/6 mice with M-CSF-expressing 3LL cells. The mice inoculated with the lowest M-CSF-producing 3LL clone showed significant prolongation of the survival compared with wild-type 3LL- Inoculated mice, and 70% or more of the mice inoculated with 3LL clones with higher M-CSF production rejected inoculation. Mice injected with radiation-inactivated M-CSF-expressing 3LL cells before or after Inoculation of wild-type 3LL cells showed prolonged survival compared with mice injected with radiated control 3LL cells before or after transplantation of wild-type cells. In vivo depletion of effector subpopulations by injection of antibodies against CD4+ T cells, CD8+ T cells, or natural killer (NK) cells suggested involvement of NK cells and CD4+ T cells in M-CSF-mediated antitumor cytotoxicity in M-CSF- producing 3LL cells-inoculated mice. Severe combined immunodeficiency (SCID) mice with defective T- and B-cell function showed prolonged survival duration after inoculation with M-CSF-expressing 3LL cells compared with those transplanted with control 3LL cells, and this effect of M-CSF expression by 3LL-cells in SCID mice was also abolished by in vivo depletion of NK cells by antibody injection. These findings together with the previous reports that M-CSF augments antibody- dependent and-independent antitumor cytotoxicity suggest that M-CSF induces tumor immunity in this cytokine-expressing tumor- transplantation model.

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