Macrophage Colony-stimulating Factor Gene Research Articles

Highly Successful Therapeutic Vaccinations Combining Dendritic Cells and Tumor Cells Secreting Granulocyte Macrophage Colony-stimulating Factor

In an attempt to induce potent immune antitumor activities, we investigated, within the rat 9L gliosarcoma model, distal therapeutic vaccinations associating three therapies: dendritic cell vaccination, intratumoral granulocyte macrophage colony-stimulating factor (GM-CSF) gene transfer, and tumor apoptosis induction. Vaccines of dendritic cells coinjected with processed GM-CSF secreting 9L cells induced systemic responses, resulting in the complete regression of distant preimplanted 9L tumor masses in, with the best strategy, 94% of male rats. All of the cured rats developed a long-term resistance to a rechallenge with parental cells. The curative responses were correlated with the detection of elevated specific cytotoxic activities and a CD4+, CD8+ T cell-, and natural killer (NK) cell-mediated IFN-gamma production. The survival rate of the rat seemed more directly linked to the amount of GM-CSF secreted by the transduced tumor cells, which in turn depended on the toxicity of the apoptosis-inducing treatment, than to the level of apoptosis induced. Unexpectedly, alive GM-CSF secreting 9L cells became apoptotic when injected in vivo. Thus we documented the positive role of apoptosis in the induction of therapeutic antitumor responses by comparing, at equal GM-CSF exogenous supply, the effects of dendritic cells coinjected with apoptotic or necrotic 9L cells. The data showed the superior therapeutic efficiency of combined vaccines containing apoptotic tumor cells. In conclusion, vaccinations with dendritic cells associated with apoptotic tumor cells secreting GM-CSF show a very high therapeutic potency that should show promise for the treatment of human cancer.

Monitoring the Response of Orthotopic Bladder Tumors to Granulocyte Macrophage Colony-Stimulating Factor Therapy Using the Prostate-Specific Antigen Gene as a Reporter

Although orthotopic animal models of cancer best reflect the disease in humans, a major drawback of these models is the inability to monitor tumor growth accurately. Our aims were to produce a bladder tumor cell line (MB49) that secreted human prostate-specific antigen (PSA), analyze the feasibility and accuracy of PSA as a biomarker for monitoring orthotopic bladder tumor growth, and evaluate the effectiveness of granulocyte macrophage colony-stimulating factor (GM-CSF) gene therapy using this model. PSA secretion was assessed after both s.c. and orthotopic implantation of MB49-PSA cells in C57BL/6 mice. PSA levels in mouse serum and urine samples were monitored at 2- to 3-day intervals by ELISA. Using the orthotopic model, mice with confirmed tumors were given liposome-mediated GM-CSF gene therapy twice a week for 3 weeks intravesically and PSA levels monitored. The MB49-PSA cells behaved similarly as the parental cell line and produced high levels of PSA both in vitro and in vivo. In the s.c. model, the level of PSA produced correlated with tumor volume (r = 0.96). In the orthotopic model, PSA could be detected in serum and urine on the fourth day after implantation. PSA levels over the treatment period indicated that tumor growth was inhibited by GM-CSF gene therapy. Up to 50% of the treated mice were cured. Cytokine array analysis revealed that GM-CSF gene therapy induced the production of other cytokines and chemokines. MB49 cells modified to secrete PSA are a reliable method to evaluate therapeutic modalities for bladder cancer.

Thyroid cancer immuno-therapy with retroviral and adenoviral vectors expressing granulocyte macrophage colony stimulating factor and interleukin-12 in a rat model.

Introduction of genes encoding immuno-stimulatory cytokine(s) into cancer cells is well known to enhance anti-tumour immunity. The present studies were designed to evaluate the therapeutic efficacy of retroviral- and adenoviral-mediated delivery of IL-12 and/or granulocyte macrophage colony-stimulating factor (GM-CSF) genes for thyroid cancer in an immuno-competent rat model. A rat thyroid cancer cell line FRTL-Tc syngeneic to Fisher rat was used. Expression of these exogenous cytokines did not affect in vitro cell growth. Subcutaneous injection of FRTL-Tc cells retrovirally transduced with IL-12 or GM-CSF genes formed significantly smaller tumours than that of the parental cells, but had little effect on growth of distant tumours, suggesting no vaccine effect. Similarly, injection of the cells infected with adenovirus expressing IL-12 or GM-CSF (AdIL-12 or AdGM-CSF) almost completely abolished tumourigenicity and injection of AdGM-CSF into pre-established tumours significantly inhibited growth of the tumours injected; neither, however, showed a systemic vaccine affect. On the other hand, injection of AdIL-12 into the pre-established tumours significantly inhibited growth of not only the tumours injected but also distant tumours, indicating induction of systemic anti-tumour immunity. Serum IL-12 was detectable only in this approach. There was neither a synergistic or additive effect of these two cytokines. Our data demonstrate in a rat thyroid cancer model that only injection of AdIL-12 into the pre-established tumours elicited systemic anti-tumour immunity, but injection of AdGM-CSF or injection of the cells expressing IL-12 or GM-CSF elicited only local effect, indicating that in situdelivery of IL-12 gene with adenovirus appears most efficacious but may still require adjuvant modalities to enhance the anti-tumour effect.

Gene expression of leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF) in bovine endometrium during early pregnancy

Leukemia inhibitory factor (LIF) and macrophage colony stimulating factor (M-CSF), members of the group of hemopoietic cytokines, play a primary role in the control of embryo development and implantation and in the growth of the placenta in humans and mice. Gene expressions of LIF and M-CSF were investigated using quantitative RT-PCR in bovine endometrial tissues during early and mid-pregnancy (Days 16–17, 20–21, 30–36, 48–49 and 74–140) and during the estrous cycle (Days 13–14). Leukemia inhibitory factor and M-CSF genes were expressed in all samples examined. Significant differences were found between the gene expression patterns of LIF and M-CSF. Leukemia inhibitory factor expression level at Days 48–49 was the highest in caruncular endometrium, however, the large variability negated any significant differences. Leukemia inhibitory factor expression levels in intercaruncular endometrium at Days 48–49 and 74–140 of pregnancy were greater than at Days 13–14 of the estrous cycle and at other days of pregnancy. No significant change was recognized in M-CSF expression levels in caruncular endometrium. Macrophage colony stimulating factor expression level in intercaruncular endometrium at Days 74–140 was greater than those of the other samples. These results suggest that LIF and M-CSF are produced in the endometrium and may play different roles in early and mid-pregnancy.

Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells

Osteoclasts are derived from hematopoietic precursor cells belonging to the monocyte/macrophage lineage. Osteoclast development has been reported to be regulated by several molecules such as macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor (NF)-kappaB ligand (RANKL), and a decoy receptor of RANKL, osteoprotegerin (OPG). Recently, it was demonstrated that the Notch signaling pathway regulates myeloid differentiation and antagonizes cell fate determination, however, the effect of Notch signaling on the osteoclast lineage has not been reported. In this study, we examined the effect of signaling via Notch receptors on the differentiation into osteoclasts by using cells from the bone marrow, spleen, and peritoneal cavity, and a cloned macrophagelike cell line. Osteoclastogenesis was inhibited by an immobilized Notch ligand, Delta-1. The dish-adherent bone marrow cells precultured with M-CSF expressed both Mac-1 and M-CSF receptors, c-Fms; osteoclastogenesis of these cells was efficiently inhibited. The immobilized Delta-1 also down-regulated the surface c-Fms expression, while the c-Fms gene expression was not changed. Genes for Notch receptors and Notch ligands are expressed in not only hematopoietic cells but also stromal cells that support osteoclast development. Constitutively active Notch1-transfected stromal cells showed increased expression of RANKL and OPG genes, and strong inhibition of M-CSF gene expression, resulting in reduction of their ability to support osteoclast development. Taken together, these findings indicate that Notch signaling affects both osteoclast precursors and stromal cells and thereby negatively regulates osteoclastogenesis.

Granulocyte macrophage-colony stimulating factor gene transfer to induce a protective anti-tumoral immune response against the 9L rat gliosarcoma model

The injection of irradiated tumor cells genetically engineered to express the granolocyte macrophage-colony stimulating factor (GM-CSF) is reported as stimulating antitumoral immunity in several animal models. We used the 9L gliosarcoma rat model to investigate the potency of this strategy in relation to the central nervous system. After in vitro transduction experiments to generate 9L murine cells producing GM-CSF (9LmGM-CSF cells), we found that with the exception of one rat subcutaneously (s.c.) grafted with 108 9LmGM-CSF cells, syngenic rats s.c. injected (dorsal route) with 106-108 viable 9LmGM-CSF cells did not develop s.c. 9L gliosarcomas, while rats s.c. grafted with the same number of naive 9L cells died between 25 and 45 days postgraft. Intracerebral stereotactic injections of 4.104 naive 9L (without s.c. 9LmGM-CSF treatment) killed all the rats within 18-24 days, while s.c. grafting with 108 9LmGM-CSF cells wholly prevented the development of 9L brain gliosarcomas. These results outline the feasibility of the GM-CSF gene transfer approach in glioma (or at least gliosarcoma) gene therapy and could therefore serve as a basis for the development of an adjuvant treatment of glioblastomas using GM-CSF-producing tumor cell vaccines.

Heat shock proteins, inflammation, and cardiovascular disease.

Atherosclerosis and its associated complications, such as coronary artery disease, peripheral vascular disease, and stroke, are the leading causes of morbidity and mortality in all racial groups of most westernized societies. A better understanding of the events that lead to the induction and progression of atherosclerosis and the development of strategies that control these processes would have a significant impact on human health. Atherosclerosis is a chronic inflammatory condition that usually begins at an early age in the absence of lipid accumulation, with fatty streaks composed of lipid-laden macrophages (foam cells) developing at later stages. T lymphocytes, predominantly of the CD4+ phenotype, are associated with the intima layer of the vessel. The inflammatory process at atherogenic sites leads to the production of cytokines and other inflammatory mediators, resulting in cell migration, proliferation, extracellular matrix production, and plaque development.1–3⇓⇓ Monocytes and macrophages appear intimately involved with the development of atherosclerosis; mice with the osteopetrotic ( op ) mutation in the macrophage colony-stimulating factor (M-CSF) gene, which results in a complete absence of M-CSF in the serum and tissues and a marked reduction in the number of circulating monocytes, exhibit significantly less atherosclerosis than control littermates when bred into an apolipoprotein (apo) E–deficient background,.4 The precise role of T and B cells in atherosclerosis remains unclear. Although activated T cells are a dominant feature of atherosclerotic lesions,5,6⇓ studies demonstrating that atherosclerosis can be induced by hypercholesterolemia in mice deficient in T and B cells suggest that these cells are not essential.7–9⇓⇓ However, the cholesterol levels in these studies far exceeded those present in human subjects without any genetic defect, and the findings should therefore be interpreted with caution. Indeed, other evidence indicates that T cells may attenuate atherogenesis by some means, because the elimination of …

Reduced tissue macrophage population in the lung by anticancer agent cyclophosphamide: restoration by local granulocyte macrophage–colony-stimulating factor gene transfer

Granulocytopenia is thought to be the sole mechanism underlying the increased susceptibility to bacterial infection in hosts with anticancer chemotherapy. Little is known about the functional state of tissue macrophage populations in such hosts. Using a model of chemotherapy-induced leukopenia, the number and function of alveolar macrophages (AMS) were examined during and after multiple injections of an anticancer agent, cyclophosphamide (CP). Although CP quickly reduced peripheral blood leukocytes, the number of these cells rebounded quickly 3 to 4 days after the withdrawal of CP. Accompanying blood leukopenia was a profound reduction in the number of AMs. Contrary to the rapid onset of blood leukopenia, tissue macrophage deficiency was a more chronic process that worsened gradually as the CP regimen continued. Of importance, in contrast to blood leukopenia, which restored itself shortly after CP withdrawal, tissue macrophage deficiency was not immediately self-recoverable in spite of a restored number of circulating leukocytes. Although AMS had a decreased ability to proliferate during, but not after, the CP regimen, these cells retained a normal ability to release tumor necrosis factor-alpha and nitric oxide. To identify the potential therapeutics for recovering macrophages, a gene vector expressing granulocyte macrophage-colony-stimulating factor (GM-CSF) was delivered either systemically or locally. GM-CSF transgene was able to expand macrophage populations only when delivered to the lung after, but not during, the CP regimen. This study thus identifies tissue macrophage deficiency as a mechanism of weakened innate immunity by chemotherapy and suggests the usefulness of topical GM-CSF transgene expression for restoring innate immunity in the lung.

Metallothionein is required for zinc-induced expression of the macrophage colony stimulating factor gene.

Macrophage colony stimulating factor (M-CSF) plays an important role in the proliferation and differentiation of mononuclear phagocytes. The present study investigates the effect of zinc on M-CSF expression in MC3T3-E1 and L929 cells. Zinc dose-dependently increased M-CSF mRNA levels. The time-course of zinc-induced M-CSF mRNA expression peaked at 6 h. Stability studies of mRNA using actinomycin D revealed that zinc does not affect M-CSF mRNA stability. We examined the function of the M-CSF gene promoter using a luciferase reporter assay. A construct containing the -467/+39 region of the promoter was upregulated by zinc. In the presence of cycloheximide, zinc did not induce a greater increase in the M-CSF mRNA than cycloheximide alone. To confirm the effect of MT on M-CSF mRNA expression, mouse lung fibroblasts (MLFs) were prepared from MT+/+ and MT-/- mice. Zinc induced an increase in the expression of M-CSF in MT+/+ MLFs, but this response was not evident in MT-/- MLFs. Moreover, overexpression of MT upregulated M-CSF mRNA expression as well as M-CSF secretion. Our findings suggest that MT expression mediates zinc regulation of M-CSF gene expression at the transcriptional level.

Oxidized low density lipoprotein inhibits macrophage apoptosis through activation of the PI 3-kinase/PKB pathway

Oxidized LDL (oxLDL) is known to induce endothelial adhesion molecule and monocyte chemoattractant protein 1 expression and this is thought to be involved in monocyte recruitment into atherosclerotic lesions. oxLDL has also been found to induce macrophage proliferation. The purpose of the present study was to determine whether oxLDL might also have the ability to increase macrophage populations by inhibiting apoptosis. We found that oxLDL caused a dose-dependent inhibition of the apoptosis that occurs in cultured bone marrow-derived macrophages after macrophage colony-stimulating factor (M-CSF) withdrawal without inducing proliferation. Incubation of macrophages with either native LDL or acetylated LDL had no effect on apoptosis. The prosurvival effect of oxLDL was not inhibited by neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, was maintained in mice homozygous for a mutation in the M-CSF gene, and was not due to other secreted cytokines or growth factors. oxLDL caused activation of the mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2) as well as protein kinase B (PKB), a target of phosphatidylinositol 3-kinase (PI 3-kinase). Furthermore, there was phosphorylation of two important prosurvival PKB targets, I-κBα(Ser-32) and Bad(Ser-136). The MEK inhibitors PD 98059 and U0126 blocked ERK1/2 activation but did not diminish survival. Conversely, the PI 3-kinase inhibitors LY 294002 and wortmannin blocked PKB activation, and the ability of oxidized LDL to promote macrophage survival. Taken together, these results indicate that oxLDL can directly activate a PI 3-kinase/PKB-dependent pathway that permits macrophage survival in the absence of growth factors. —Hundal, R. S., B. S. Salh, J. W. Schrader, A. Gómez-Muñoz, V. Duronio, and U. P. Steinbrecher. Oxidized low density lipoprotein inhibits macrophage apoptosis through activation of the PI 3-kinase/PKB pathway.

An Alternative Open Reading Frame of the Human Macrophage Colony-Stimulating Factor Gene Is Independently Translated and Codes for an Antigenic Peptide of 14 Amino Acids Recognized by Tumor-Infiltrating Cd8 T Lymphocytes

We show that cytotoxic T lymphocytes (CTLs) infiltrating a kidney tumor recognize a peptide encoded by an alternative open reading frame (ORF) of the macrophage colony-stimulating factor (M-CSF) gene. Remarkably, this alternative ORF, which is translated in many tumors concurrently with the major ORF, is also translated in some tissues that do not produce M-CSF, such as liver and kidney. Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected. The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues. Peptide elution indicated that tumor cells naturally present this 14 mer, which is the longest peptide known to be recognized by CTLs. Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.

CAMP attenuates interleukin-1-stimulated macrophage colony-stimulating factor (M-CSF) expression

Macrophage colony-stimulating factor (M-CSF) is a multifunctional cytokine attributed with key biological functions beyond the first discovered role in promoting proliferation of myeloid cell lineage. The human pancreatic cancer cell line MIA PaCa-2, from which the M-CSF gene was originally cloned, was used to study regulation of M-CSF expression. Expression of M-CSF was inducible by interleukin-1alpha (IL-1alpha), lipopolysaccharide (LPS) and PMA as demonstrated by a biological activity assay, Northern-blot analysis and reverse transcriptase (RT) PCR. Treatment of the cells with forskolin or dibutyryl-cAMP attenuated the expression of M-CSF induced by IL-1alpha or LPS, but not by PMA. Electromobility shift assays showed that IL-1alpha predominantly activated nuclear factor kappaB (NF-kappaB), while PMA preferentially activated activator protein-1 (AP-1). The activation of NF-kappaB, but not AP-1, could be attenuated by cAMP elevation. Relative RT-PCR demonstrated that the expression of a 1.6-kb M-CSF mRNA transcript was more effectively induced by IL-1alpha than a 4.0-kb transcript. By and large the induced expression of both mRNA transcripts could be attenuated by cAMP. M-CSF promoter-driven luciferase reporter-gene assays revealed that cAMP elevation attenuated the IL-1-induced transcription activation of the M-CSF promoter, but it had no effect on PMA-induced transcription. Our findings suggest that cAMP regulates M-CSF gene expression at the transcriptional level and that its inhibitory effect involves NF-kappaB signalling pathway.

Granulocyte–Macrophage Colony-Stimulating Factor Gene Transfer to Dendritic Cells or Epidermal Cells Augments Their Antigen-Presenting Function Including Induction of Anti-Tumor Immunity

Dendritic antigen-presenting cells derived from epidermis (Langerhans cells), bone marrow, and peripheral blood can present a wide variety of antigens, including tumor-associated antigens, for various immune responses. The development and function of dendritic cells is dependent upon a number of cytokines including granulocyte-macrophage-colony-stimulating factor. For example, Langerhans cells can present tumor-associated antigens for the induction of substantial in vivo anti-tumor immunity but only after activation in vitro by granulocyte-macrophage-colony-stimulating factor. Thus, we reasoned that insertion of a cDNA for granulocyte-macrophage-colony-stimulating factor into dendritic antigen-presenting cells may allow for autocrine stimulation and increased antigen-presenting capability. To test this possibility, we utilized an adenovirus vector to insert a cDNA for murine granulocyte-macrophage-colony-stimulating factor into the dendritic cell lines XS52-4D and XS106 (derived from neonatal mouse epidermis), bone marrow-derived dendritic cells, and epidermal cells that contain Langerhans cells. Infection of each of these cell types resulted in release of abundant quantities of granulocyte-macrophage-colony-stimulating factor. XS52-4D and XS106 cells infected with adenovirus granulocyte-macrophage-colony-stimulating factor exhibited prolonged dendrites and greater expression of major histocompatibility complex class II molecules and CD86 compared with cells infected with a null vector. Granulocyte-macrophage-colony-stimulating factor cDNA-containing XS cells, bone marrow-derived dendritic cells, and epidermal cells had more potent alloantigen presenting capability than cells infected with a null vector. Most importantly, granulocyte-macrophage-colony-stimulating factor gene-transferred epidermal cells were able to present tumor-associated antigens for in vivo anti-tumor immunity against challenge with the S1509a spindle-cell tumor whereas null vector-infected cells were unable to prime for immunity. These results suggest that introduction of a cDNA for granulocyte-macrophage-colony-stimulating factor into dendritic cells may be an effective means to augment their antigen-presenting capability and that granulocyte-macrophage-colony-stimulating factor gene-transfer- red epidermal cells may be useful in tumor vaccination strategies.

Combined therapy with anti-P-glycoprotein antibody and macrophage colony-stimulating factor gene transduction for multiorgan metastases of multidrug-resistant human small cell lung cancer in NK cell-depleted SCID mice.

Our aim was to determine the antimetastatic potential of anti-P-glycoprotein (P-gp) antibodies (Abs) against multidrug-resistant (MDR) human small cell lung cancer (SCLC) cells expressing P-gp. Human SCLC cells H69 (P-gp negative) and its etoposide-resistant variant H69/YP (P-gp positive) were used. H69 and H69/VP cells injected i.v. metastasized to the liver, kidneys and systemic lymph nodes of NK cell-depleted severe combined immunodeficient (SCID) mice. H69/VP cells, but not H69 cells, were resistant to treatments with vindesine. Treatment with mouse-human chimeric anti-P-gp Ab (MH162) and its mouse counterpart (MRK-16) reduced metastasis of H69/VP cells in various organs and prolonged the survival of tumor-bearing mice, although they were less effective if injected at late times (after 28 days). Treatment with another mouse anti-Pgp Ab, MRK-17, was effective only against liver metastasis. MH162 and MRK-16 efficiently induced Ab-dependent cellular cytotoxicity (ADCC) by peritoneal macrophages against H69/VP cells in vitro, but MRK-17 was less effective, in accordance with their in vivo antimetastatic potential. Gene transfection of macrophage colony-stimulating factor (M-CSF) into H69/VP cells to augment macrophage-mediated ADCC resulted in inhibition of metastasis to the liver and lymph nodes, but not kidneys. Combined treatment with a low dose of MRK-16 completely cured metastasis of M-CSF transfectant, but not of the mock transfectant. Our findings suggest that while anti-P-gp Abs had antimetastatic potential against SCLC cells expressing P-gp, combined treatment with M-CSF gene transduction to augment the therapeutic efficacy of anti-P-gp Abs may be beneficial for eradicating metastatic MDR SCLC in humans.

The zinc finger protein A20 inhibits TNF-induced NF-kappaB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-kappaB-inhibiting protein ABIN.

The zinc finger protein A20 is a tumor necrosis factor (TNF)- and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-kappaB)-dependent gene expression. However, the molecular mechanism by which A20 exerts this effect is still unclear. We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-kappaB, although it completely prevents the TNF- induced activation of an NF-kappaB-dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage-colony stimulating factor gene expression. Moreover, NF-kappaB activation induced by overexpression of the TNF receptor-associated proteins TNF receptor-associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor-associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-kappaB activation induced by overexpression of NF-kappaB-inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected. These results demonstrate that A20 inhibits NF-kappaB-dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK-IkappaB kinase pathway and that is specifically involved in the transactivation of NF-kappaB. Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-kappaB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-kappaB inhibiting protein, ABIN.

Heterogeneity of multiorgan metastases of human lung cancer cells genetically engineered to produce cytokines and reversal using chimeric monoclonal antibodies in natural killer cell-depleted severe combined immunodeficient mice.

Lung cancer is a major cause of cancer deaths, most of which can be attributed to distant multiorgan metastases. To examine the cellular and molecular mechanisms of lung cancer metastasis to distant organs, we have established novel models of human lung cancer (small cell and non-small cell lung cancer) metastasis in natural killer cell-depleted severe combined immunodeficient (SCID) mice. We investigated whether local production of the cytokines responsible for regulation of macrophage function at tumor growth sites affects the pattern of lung cancer metastasis in distant organs. Several lung cancer cell lines were genetically engineered to produce human macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein-1 (MCP-1), and their metastatic potentials were assessed. Interestingly, M-CSF gene transduction had an antimetastatic effect for the liver and lymph nodes, but not the kidneys. In contrast, MCP-1 gene-modified lung cancer cells and their parent cells had identical metastatic potentials. These findings indicate a possible role for cytokines and suggest that lung cancer has metastatic heterogeneity. Examining ways of controlling human lung cancer metastases, we investigated the antimetastatic effect of chimeric monoclonal antibodies (MAbs) against P-glycoprotein and ganglioside GM2 (MH162 and KM966, respectively). Both MAbs, when given on days 2 and 7, inhibited the development of distant metastases of lung cancer in a dose-dependent fashion. Combined use of anti-P-glycoprotein MAb with M-CSF or MCP-1 gene transduction caused complete inhibition of metastasis of H69/VP cells. The antimetastatic effect of these MAbs in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity reaction mediated by mouse macrophages. These findings suggest that the mouse-human chimeric MAb in combination with cytokine gene transduction may be useful for the eradication of lung cancer metastases in humans.

Enhanced cellular immunity to hepatitis C virus nonstructural proteins by codelivery of granulocyte macrophage-colony stimulating factor gene in intramuscular DNA immunization

Hepatitis C virus (HCV) nonstructural (NS) proteins appeared to be important targets for HCV vaccine development, since NS-specific T-helper-cell responses are associated with clearance from acute HCV infection. In this report, we have constructed a plasmid, pTV–NS345, that encodes the HCV NS3, NS4 and NS5 proteins (NS345) and a bicistronic plasmid, PTV–NS345/GMCSF, in which the HCV NS345 polyprotein and GMCSF are translated independently. Intramuscular inoculation with pTV–NS345 plasmid DNA into the Buffalo rats generated both antibody and T-cell proliferative responses to each NS protein. The expression of GMCSF, together with HCV NS345 proteins, appeared to significantly increase T-cell proliferative responses. In particular, the inoculation of a bicistronic plasmid generated higher T-cell proliferative responses to each NS protein than did the coinjection of two separate plasmids, pTV–NS345 and pTV-GMCSF. These results demonstrate that the codelivery of GMCSF augmented HCV NS345-specific cellular immunity and that the intensity of the immunity was differed depending on how GMCSF gene is codelivered.

Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1.

Central to the pathogenesis of osteoporosis is the ability of estrogen deficiency to increase osteoclast formation by enhancing stromal cell production of the osteoclastogenic cytokine macrophage colony-stimulating factor (M-CSF). We report that stromal cells from ovariectomized mice exhibit increased casein kinase II-dependent phosphorylation of the nuclear protein Egr-1. Phosphorylated Egr-1 binds less avidly to the transcriptional activator Sp-1 and the resulting higher levels of free Sp-1 stimulate transactivation of the M-CSF gene. Estrogen replacement fails to block M-CSF mRNA expression and osteoclast formation in ovariectomized mice lacking Egr-1, confirming the critical role played by this transcription factor in mediating the antiosteoclastogenic effects of estrogen. Thus, by downregulating formation of a novel Egr-1/Sp-1 complex in stromal cells, estrogen deficiency results in enhanced levels of free Sp-1 and increased M-CSF gene expression and osteoclast formation.

PU.1 regulates both cytokine-dependent proliferation and differentiation of granulocyte/macrophage progenitors.

PU.1 is a unique regulatory protein required for the generation of both the innate and the adaptive immune system. It functions exclusively in a cell-intrinsic manner to control the development of granulocytes, macrophages, and B and T lymphocytes. We demonstrate that mutation of the PU.1 gene causes a severe reduction in myeloid (granulocyte/macrophage) progenitors. PU.1 -/- myeloid progenitors can proliferate in vitro in response to the multilineage cytokines interleukin-3 (IL-3), IL-6 and stem cell factor but are unresponsive to the myeloid-specific cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and M-CSF. The failure of PU.1 -/- progenitors to respond to G-CSF is bypassed by transient signaling with IL-3. In the presence of IL-3 and G-CSF, PU.1 -/- progenitors can differentiate into granulocytic precursors containing myeloperoxidase-positive granules. Thus PU.1 is not essential for specification of granulocytic precursors, but is required for their further differentiation. The failure of PU.1 -/- progenitors to respond to M-CSF is due to lack of c-fms gene transcription. Transduction of c-fms into PU.1 -/- myeloid progenitors bypasses the block to M-CSF-dependent proliferation but does not induce detectable macrophage differentiation. Therefore, PU. 1 appears to be essential for specification of monocytic precursors. Importantly, retroviral transduction of PU.1 into mutant progenitors restores responsiveness to myeloid-specific cytokines and development of mature granulocytes and macrophages. Thus PU.1 controls myelopoiesis by regulating both proliferation and differentiation pathways.

Cellular and molecular characteristics of inflammation in chronic bronchitis.

To examine the inflammatory process in chronic bronchitis, we evaluated the cell and cytokine profile in bronchoalveolar lavage fluid from 12 chronic bronchitis patients who smoked, six chronic bronchitis patients who did not smoke, 10 subjects control subjects without pulmonary diseases who smoked and eight control subjects who did not smoke. Chronic bronchitis patients who smoked had increased numbers of macrophages, neutrophils, eosinophils, mast cells and activated CD8+ T lymphocytes and predominantly expressed interleukin-8, tumour necrosis factor alpha and interleukin-2 genes and proteins. The number of macrophages and neutrophils and the expression of interleukin 8 were also increased in control subjects who smoked compared with healthy subjects who did not smoke. Chronic bronchitis patients who did not smoke had increased numbers of eosinophils, mast cells and activated CD4+ T lymphocytes and predominantly expressed interleukin-5 and granulocyte-macrophage colony-stimulating factor genes and proteins. Thus, the cellular and molecular characteristics of the inflammatory process in chronic bronchitis patients who smoke and do not smoke are different, suggesting a different pathogenesis of the disease.