L929-conditioned Medium Research Articles

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages.

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

Generation of a 3D melanoma model and visualization of doxorubicin uptake by fluorescence imaging.

Melanoma is the most dangerous type of skin cancer and is responsible for 75% of deaths from skin cancers. For an accurate evaluation of potential treatment efficacy, it is important to use study models as close as possible to the in vivo conditions. A 3D model consisting of B16F10 spheroids was developed using liquid overlay technique on plates coated with 1% agarose, in the presence of 1% methylcellulose and L929-conditioned medium. The model is suitable and can be further used for more complex in vitro drug testing than the classical 2D approach. For exemplification, the behavior of a well-known cytostatic, doxorubicin (DOX), was evaluated in spheroids as compared to classical 2D culture conditions. Fluorescence imaging was used to visualize DOX uptake by B16F10 spheroids at different periods of time. The results showed that a much higher DOX concentration is necessary to produce similar effects compared with the monolayer. The fluorescence images revealed that at least 4 h of stimulation is needed for a sufficient DOX uptake. The 3D model developed in this study was suitable to investigate drug penetration in time. Our findings may explain the decrease of the doxorubicin therapeutical effect, suggesting the need of maintaining the drug concentration at the tumoral place for at least 2 h upon administration. Similar or more advanced studies can lead to a better understanding of drug delivery kinetics and distribution upon administration, conducing toward a better performance in designing suitable delivery systems for obtaining the optimum dose-response effect.

Toll-like Receptor 3 Is Required for Development of Retinopathy Caused by Impaired All-trans-retinal Clearance in Mice

Chronic inflammation is an important component that contributes to many age-related neurodegenerative diseases, including macular degeneration. Here, we report a role for toll-like receptor 3 (TLR3) in cone-rod dystrophy (CORD) of mice lacking ATP-binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8), proteins critical for all-trans-retinal clearance in the retina. Increased expression of toll-like receptor-signaling elements and inflammatory changes were observed in Rdh8(-/-)Abca4(-/-) eyes by RNA expression analysis. Unlike 3-month-old Rdh8(-/-)Abca4(-/-) mice that developed CORD, 6-month-old Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice did not evidence an abnormal retinal phenotype. Light-induced retinal degeneration in Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice was milder than that in Rdh8(-/-)Abca4(-/-) mice, and a 2-fold increased TLR3 expression was detected in light-illuminated retinas of Rdh8(-/-)Abca4(-/-) mice compared with nonilluminated retinas. Poly(I-C), a TLR3 ligand, caused caspase-8-independent cellular apoptosis. Whereas poly(I-C) induced retinal cell death in Rdh8(-/-)Abca4(-/-) and WT mice both in vivo and ex vivo, this was not seen in mice lacking Tlr3. Far fewer invasive macrophage/microglial cells in the subretinal space and weaker activation of Muller glial cells were exhibited by Tlr3(-/-)Rdh8(-/-) Abca4(-/-) mice compared with Rdh8(-/-)Abca4(-/-) mice at 3 and 6 months of age, indicating that loss of TLR3 inhibits local inflammation in the retina. Both poly(I-C) and endogenous products emanating from dying/dead retinal cells induced NF-κB and IRF3 activation. These findings demonstrate that endogenous products from degenerating retina stimulate TLR3 that causes cellular apoptosis and retinal inflammation and that loss of TLR3 protects mice from CORD.

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation

Lipopolysaccharide (LPS) activates innate immune responses through TLR4.MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4.MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV(A), a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV(A) activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV(A) activation. Computational studies suggested that unique ionic interactions exist between lipid IV(A) and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4'-phosphate on lipid IV(A) interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys(367) and Arg(434)) at the dimerization interface. When replaced with their negatively charged human counterparts Glu(369) and Gln(436), mouse TLR4 was no longer responsive to lipid IV(A). In contrast, human TLR4 gained lipid IV(A) responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV(A) species specificity. Thus, using lipid IV(A) as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.

Bone Marrow-Derived Macrophages (BMM): Isolation and Applications

INTRODUCTIONBone marrow-derived macrophages (BMM) are primary macrophage cells, derived from bone marrow cells in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is a lineage-specific growth factor that is responsible for the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Mice lacking functional M-CSF are deficient in macrophages and osteoclasts and suffer from osteopetrosis. In this protocol, bone marrow cells are grown in culture dishes in the presence of M-CSF, which is secreted by L929 cells and is used in the form of L929-conditioned medium. Under these conditions, the bone marrow monocyte/macrophage progenitors will proliferate and differentiate into a homogenous population of mature BMMs. The efficiency of the differentiation is assessed using fluorescence-activated cell sorting (FACS) analysis of Mac-1 and 4/80 surface antigen expression. Once differentiated, the BMMs are suitable for numerous types of experimental manipulations, including morphological, gene expression, and physiological studies. For example, phagocytic cells such as macrophages have a unique ability to ingest microbes. We describe a test for the phagocytic efficiency of BMMs by exposing them to fluorescently labeled yeast zymosan bioparticles. Also, a method to deliver DNA or small interfering RNAs (siRNAs) into these hard-to-transfect cells is described. Finally, the proliferation of the BMMs is assayed using carboxyfluorescein succinimidyl ester (CFSE), a fluorescein derivative that partitions equally between daughter cells after cell division.

Methods of in vitro macrophage maturation confer variable inflammatory responses in association with altered expression of cell surface dectin-1

Macrophage differentiation and polarization occur in vivo under the influence of the localized cytokine milieu. In vitro studies frequently rely on cellular differentiation in culture; hence, unrecognized variables could have a large influence on the observed cellular phenotype. We measured macrophage in vitro responses to fungal ligands (Aspergillus germ tubes and zymosan), focusing on the degree to which culture conditions impact stimulatory responses through the C-type lectin receptor, dectin-1, which is involved in both MyD88-dependent and MyD88-independent signaling in response to fungal β1,3 glucan. Results show that macrophages harvested from different murine anatomic sites exhibit varying degrees of MyD88-dependence, with bone marrow-derived macrophages (BMDM) cultured in L929 conditioned medium (L929 CM) exhibiting the largest degree of MyD88-independence. After differentiation in recombinant MCSF (rMCSF), MyD88−/− macrophages have decreased surface expression of dectin-1 compared to wild type macrophages; however, culture in L929CM results in higher, and equivalent expression of dectin-1 on both MyD88−/− and wild type BMDM. In addition to MCSF, L929CM contains high amounts of VEGF, MCP-1, KC, and MIG, and low amounts of FGF-β, Eotaxin, IL-10, IL-9, and IL-12. Thus, methods of in vitro maturation dictate variable inflammatory responses by MyD88−/− macrophages in association with altered expression of cell surface dectin-1. L929 conditioned medium is a suboptimal alternative to rMCSF for in vitro studies. As MyD88−/− BMDM exhibit low surface expression of dectin-1 after in vitro culture in rMCSF, differences in dectin-1 dependent, MyD88-independent signaling may account for some of the phenotypes currently ascribed to MyD88-deficiency alone.

Effect of alcoholic extract of Ayurvedic herb Tinospora cordifolia on the proliferation and myeloid differentiation of bone marrow precursor cells in a tumor-bearing host

The present study investigates the effect of in vivo administration of alcoholic extract of Tinospora cordifolia whole plant (ALTC) on the proliferation and myeloid differentiation of bone marrow hematopoietic precursor cells in mice bearing a transplantable T cell lymphoma of spontaneous origin designated as Dalton's lymphoma (DL). BMC obtained from ALTC administered DL-bearing mice showed an enhanced BMC proliferation and colony forming ability in vitro in response to L929 conditioned medium as a source of colony stimulating factor (CSF). The number of granulocyte–macrophages colony (CFU-GM) was predominantly higher in the cultures of BMC obtained from ALTC administered mice as compared to mice injected with PBS alone. An increase in the count of bone marrow derived macrophages (BMDM) from ALTC administered mice was also observed along with an increase in the count of tumor associated macrophages. The BMDM obtained from ALTC administered mice showed an enhanced response to signal of LPS for activation to produce IL-1 and TNF. This study indicates that the T. cordifolia can influence the myeloid differentiation of bone marrow progenitor cells and the recruitment of macrophages in response to tumor growth in situ.

Abca7 Null Mice Retain Normal Macrophage Phosphatidylcholine and Cholesterol Efflux Activity despite Alterations in Adipose Mass and Serum Cholesterol Levels

Mutations in the A class of ATP-binding cassette transporters (ABCA) are causally implicated in three human diseases: Tangier disease (ABCA1), Stargadt's macular degeneration (ABCA4), and neonatal respiratory failure (ABCA3). Both ABCA1 and ABCA4 have been shown to transport lipids across cellular membranes, and ABCA3 may play a similar role in transporting pulmonary surfactant. Although the functions of the other 10 ABCA class transporters identified in the human genome remain obscure, ABCA7-transfected cells have been shown to efflux lipids in response to stimulation by apolipoprotein A-I. In an effort to elucidate the physiologic role of ABCA7, we generated mice lacking this transporter (Abca7-/- mice). Homozygous null mice were produced from intercrosses of heterozygous null mice at the expected Mendelian frequency and developed normally without any obvious phenotypic abnormalities. Cholesterol and phospholipid efflux stimulated by apolipoprotein A-I from macrophages isolated from wild type and Abca7-/- mice did not differ, suggesting that these activities may not be central to the physiological role of the transporter in vivo. Abca7-/- females, but not males, had significantly less visceral fat and lower total serum and high density lipoprotein cholesterol levels than wild type, gender-matched littermates. ABCA7 expression was detected in hippocampal and cortical neurons by in situ hybridization and in brain and white adipose tissue by Western blotting. Induction of adipocyte differentiation from 3T3 fibroblasts in culture led to a marked increase in ABCA7 expression. These studies suggest that ABCA7 plays a novel role in lipid and fat metabolism that Abca7-/- mice can be used to elucidate.

Enhanced cloning efficiency of mouse bone marrow macrophage progenitors correlates with increased content of CSF-1 receptor of their progeny at low oxygen tension

Mononuclear phagocytes are located in every tissue of metazoan organisms. In this extravascular space, they are designated as macrophages and are known to sense and process many signals including the local oxygen tension (PO 2), which ranges from 150 mmHg at the lung apices to around 40 mmHg in mixed venous blood and most organs, and to less than 10 mmHg in tissues where long-term and dynamic remodeling processes occur. Most tissue macrophages survive and maintain their differentiated status within an environment bathed by colony-stimulating factor (CSF)-1 through the CSF-1 receptor, encoded by the Csf1r gene. In order to investigate the mRNA expression profile of macrophages as a function of PO 2, we developed an in vitro model in which monocyte-derived macrophages were generated from mouse bone marrow progenitor cells grown and maintained under low (36 mmHg) or atmospheric (142 mmHg) PO 2, in the presence of L929-conditioned medium (L-CM) as a source of CSF-1. We show that CSF-1-reactive C57BL/6 bone marrow cells displayed an increased cloning efficiency under a PO 2 of 36, compared with 142 mmHg. Furthermore, we provide evidence of the overexpression of both CSF-1 receptor protein and mRNA by mouse monocyte-derived macrophages generated from bone marrow under low PO 2.

Loss of growth factor sensitivity and development of mitoxantrone resistance in transplanted recurrent SA7HD myeloid leukemia.

Recurrent SA7HD leukemia cells have also "lost" the response to stimulation by L-929-conditioned medium (L-929-CM) and indeed were significantly insensitive to proliferative stimulation by combinations of WEHI-conditioned medium (WEHI-CM) and L-929-CM (P = 0.04) in contrast to untreated SA7HD leukemia, which displayed dose-dependent stimulation by L-929-CM and exhibited synergistic proliferative response to combinations of the two growth factors. This altered growth-factor sensitivity, which was a function of both the route of leukemia inoculation and drug administration, was detected by both the colony and the tritiated thymidine (3HTdR) incorporation assays. In contrast to transplant in normal mice, recurrent leukemia transplanted in mitoxantrone pretreated mice remained growth-factor insensitive. In addition, no growth delay of the recurrent leukemia occurred during this passage in pretreated mice. However, recovery of growth-factor sensitivity still occurred after a single subsequent transplant in normal mice. The magnitude of resistance developed by the recurrent leukemia was not increased by passage in mitoxantrone pretreated mice. Rather, the extent of resistance was a function of the dose of mitoxantrone employed in the treatment of the leukemia before recurrence. These data interpreted along with previously published observations suggest that the development of altered growth-factor sensitivity by SA7HD recurrent leukemia was directly linked to the presence of mitoxantrone. In addition, this phenomenon possibly confers survival advantage to the leukemia cells and this probably accounts for the observation that most of the drug-treated mice ultimately developed recurrent disease despite adequate treatment.

In vitro generation of equine osteoclasts from bone marrow cells using a novel culture system

We report on preliminary results of a novel in vitro culture system designed to generate equine osteoclasts in large numbers. Osteoclast generation, as determined by the expression of tartrate resistant acid phosphatase (TRAP) and ability to resorb bone, was enhanced in equine bone marrow cultures supplemented with fibroblastic cell (L929) conditioned medium (L929-CM). Bone marrow was collected from a total of 12 horses and ponies and TRAP-positive cells with bone resorbing ability were generated in significant numbers in the last seven. TRAP-positive mononuclear cells appeared after three days in culture and steadily increased in number and size up to seven days when multinuclear TRAP-positive cells started to appear. L929-CM caused a significant increase in the number of TRAP-positive mononuclear cells generated after four and seven days, after which time the number of TRAP-positive cells decreased. The development of this in vitro system provides a reproducible means of generating large numbers of equine osteoclasts necessary for the study of bone remodelling and endochondral ossification in the horse.

A sequential culture approach to study osteoclast differentiation from nonadherent porcine bone marrow cells.

A "sequential culture step" system was devised to study osteoclast differentiation from newborn porcine bone marrow cells. Nonadherent cells were collected from cultures of bone marrow cells, and subsequently precultured at a low cell density in low-serum medium supplemented with L929-conditioned medium (L9-CM) derived M-CSF/CSF-1. After 4 d, adherent cells mainly composed of M-CSF-dependent macrophage/osteoclast progenitors, but devoid of stromal-like cells, were further cultured in medium supplemented with L9-CM and CM derived from serum-free cultures of fetal rat calvarial bones. This phase was characterized by a rapid induction of mono- and multinucleated (pre)osteoclast-like cells, positive for cytochemical TRAP activity, but negative for nonspecific esterase (NSE) staining. The presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] stimulated osteoclast generation, whereas calcitonin treatment significantly inhibited this process. The osteoclastic nature of the cells was confirmed by the occurrence of extensive, characteristic bone resorption on dentin slices, which was associated with release of type I collagen N-telopeptides from the bone matrix into the culture medium. The presence of a DNA synthesis inhibitor (HU) during the first 3 d of culture completely inhibited osteoclast formation, whereas HU treatment during the last phase did not affect production of multinucleated osteoclast-like cells. Likewise, a specific antibody directed against M-CSF during the first preculture period, completely abolished osteoclast formation. Adding the antibody during the last phase of the culture, however, strongly inhibited multinucleated osteoclast formation, accompanied by a significant increase in a mononuclear TRAP-positive, NSE-positive (osteoclast precursor) cell fraction. These results indicate that M-CSF is essential for progenitor proliferation as well as for (pre)osteoclast maturation and/ or fusion into multinucleated cells, but also suggest that additional soluble (bone-derived) factors are involved as cofactors in the differentiation process to committed mononuclear osteoclast precursors. The porcine marrow culture approach provides a suitable model system to investigate specific soluble osteoclast-inducing factors affecting different stages of osteoclast development.

Choosing and preparing antigen-presenting cells.

The first issue in many antigen-processing experiments is to choose and obtain appropriate antigen-presenting cells (APCs). Macrophages have characteristics that dictate their use in certain antigen-processing experiments, so this unit describes preparation of activated macrophages, which involves eliciting peritoneal macrophages from mice by infection with Listeria monocytogenes. This method is often preferred because the resulting macrophages are more consistently activated to a high degree, but macrophages can also be obtained (as described here) by sequential injection of L. moncytogenes and proteose peptone, by induction with concanavalin A (Con A), or by isolation from bone marrow. Lipopolysaccharide-stimulated B lymphoblasts can also be used as APCs. Protocols are included for preparation of L. moncytogenes stocks and preparation of L929-conditioned medium which is used for culturing bone marrow-derived macrophages.

Effect of cisplatin administration on the proliferation and differentiation of bone marrow cells of tumour-bearing mice.

In the present study the effect of tumour growth with respect to two tumour systems (Dalton's lymphoma [DL] and P815) on the in vitro, colony-forming ability (CFA) and proliferation of the bone marrow cells (BMC) of C3H/He mice was investigated. The P815-bearing mice showed an enhanced bone marrow colony forming ability in vitro, in response to L929 conditioned medium (L929 CM) used as a source of CSF. On the other hand, DL-bearing mice did not have any significant alteration in this process compared to normal mice. In vivo, administration of cisplatin resulted in a significant rise in the CFA of normal as well as DL- or P815-bearing mice. The tumour-conditioned medium (TCM) and ascitic fluid (AF) of P815 but not of DL, was found to support the in vitro colony formation of BMC obtained from cisplatin-treated or untreated mice. The number of CFU-granulocyte-macrophage was predominantly high in the cultures of BMC incubated with TCM or AF of P815. The TCM and AF of P815 also enhanced the in vitro proliferation of BMC obtained from cisplatin-treated or untreated mice. In vivo, administration of cisplatin in normal mice resulted in enhanced numbers of peritoneal exudate macrophages (PEM) and PBL. Similarly, the number of PEM and PBL was augmented in both the P815- and DL-bearing mice. However, cisplatin administration in the tumour-bearing mice decreased the count of PEM, while the number of leucocytes remain unaffected. This study indicates that the cancer chemotherapeutic drug cisplatin can influence the differentiation of the bone marrow progenitor cells in response to tumour growth in situ.

Proliferation of rat liver macrophages in vitro : Influence of hemopoietic growth factors

We examined the effects of several hemopoietic growth factors on proliferation of rat liver macrophages in vitro. The proliferative response of liver macrophages to hemopoietic growth factors was assayed on the basis of [methyl-3H]thymidine uptake. Macrophage colony-stimulating factor and recombinant murine granulocyte-macrophage colony-stimulating factor stimulated [methyl-3H]thymidine incorporation in a concentration-dependent manner. With granulocyte-macrophage colony-stimulating factor, maximum incorporation was observed at 50 U/ml, whereas with macrophage colony-stimulating factor no incorporation plateau was observed up to 50% L929-conditioned medium. Incubation of liver macrophages with various concentrations of recombinant human interleukin-2, recombinant murine interleukin-3 and recombinant human interleukin-6 or culture medium alone did not result in significant incorporation of [methyl-3H]thymidine. When liver macrophages were fractionated according to cell size, highest incorporation was observed in the large macrophages. Proliferating cells in cultures of all subfractions were microscopically identified as typical macrophages by the use of macrophage-specific monoclonal antibodies. After 6 days in culture, these macrophages had functional properties similar to those of resident liver macrophages with respect to phagocytosis and in vitro activation with immunomodulators to tumorcytotoxicity and secretion of nitric oxide and tumor necrosis factor-alpha. These results suggest that macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor play important roles among the regulatory factors that support local proliferation of rat liver macrophages.

Tumor cytostasis mediated by a monoclonal IgM antibody promoting adhesion between macrophages and tumor cells. Evidence for a lectin-like behavior.

We have shown previously that an IgM mAb (A10) recognizing Ehrlich tumor (ET) cell surface carbohydrates, inhibits in vivo ET growth by a macrophage-dependent mechanism. The inhibition mechanism involving both IgM and macrophages was unclear because receptors for IgM on macrophages are controversial and another monoclonal IgM (E1), also recognizing ET cell surface carbohydrates, was completely unable to show any protective effect. Here we show that A10, but not E1, was able to promote adhesion between macrophages and ET cells by a receptor for IgM-independent mechanism. Immunofluorescence studies showed that A10, but not E1, did react with macrophages if these cells were preincubated with a source of Ag spontaneously released from ET cells. This Ag release appeared to be required for A10-mediated adhesion, because adhesion was not obtained when ET cells fixed with paraformaldehyde were used. Cytostasis studies performed with macrophages stimulated with L-929 conditioned medium and ET cells showed that A10, but not E1 nor unrelated IgM, was able to inhibit ET cell proliferation in vitro by a mechanism involving cell contact between both cell populations. Therefore, IgM inhibition of ET growth, both in vivo and in vitro, could be explained by a lectin-like mechanism, where IgM, recognizing Ag of tumor origin, bridges macrophages to tumor cells.

Increase in Colony Stimulating Factor(CSF) in serum and augmentation of CSF responsiveness of lymphoid mononuclear cells by acute Trypanosoma cruzi infection in mice.

Acute infection of Trypanosoma (T.) cruzi to C3H/HeN mice caused the induction of a higher level of serum colony stimulating factor (CSF) activity to support the proliferation of mouse bone marrow cells. The CSF activity reached a maximum 2 days after the infection and declined thereafter. Spleen cells of the T. cruzi-infected mice showed higher levels of responsiveness to CSF in L929-conditioned medium, mouse recombinant GM-CSF and infected mouse sera as compared with normal mouse spleen cells. The induction of CSF-responding cells became plateau 4 days after the infection and it decreased thereafter. In concomitant with the production of CSF activity in the infected mouse sera, large granular cells bearing high intensity of Mac-2 antigen increased in the infected mouse spleen. These cells were nylon nonadherent and displayed inhibitory effect on T cell response to Con A. These findings indicate that T. cruzi infection induces augmentation of in vivo CSF production, leading to the abnormal proliferation of CSF-responding cells and that augmented production of, and responsiveness to, CSF might be one of important mechanisms responsible for the induction of immune abnormalities in T. cruzi-infected mice.

Formosanin-C, an immunomodulator with antitumor activity

Paris formosana Hayata (Liliaceae) grown in the mountain areas of Taiwan, has been used as a folk remedy for snake bite, and as an anti-inflammatory or anti-neoplastic agent. The effects of formosanin-C, a diosgenin saponin isolated from Paris formosana, on immune responses and transplantable murine tumor were studied. In culture systems, formosanin-C (0.03–0.16 μM) displayed significant enhancement of the blastogenic response of human peripheral blood cells to phytohemagglutinin. Formosanin-C also significantly increased the 3H-thymidine incorporation of ConA-stimulated lymphocytes at concentrations of 0.01 and 0.01 μM. The responsiveness of the granulocyte/macrophage colony-forming cells (GM-CFC) to mouse fibroblast cells L929 conditioned medium was altered in the presence of 0.01 and 0.001 μM of formosanin-C. In addition, formosanin-C given intraperitoneally activated natural killer cell activity at doses of 1–2.5 mg/kg. An intraperitoneal injection of 2.5 mg/kg of formosanin-C markedly induced interferon production, the peak blood level of which was observed 24 h after formosanin-C injection. Growth of subcutaneously transplanted MH134 mouse hepatoma was retarded by intraperitoneal treatment with 1–2.5 mg/kg of formosanin-C. The activity of 5-fluorouracil against MH-134 mouse hepatoma was potentiated by intraperitoneal treatment with formosanin-C. These results suggest that formosanin-C might display antitumor activity in association with modification of the immune system.