Productive interactions between B and natural killer cells

In this report, we have established that natural killer (NK) cells can increase IgG2a secretion by B lymphocytes as well as alter the distribution of the remaining immunoglobulin isotypes. The effect of NK cells on B cell differentiation is similar to that obtained by the direct addition of recombinant interferon-gamma (IFN-gamma) and, therefore, most likely results from the elaboration of IFN-gamma by NK cells, this is a clear demonstration that NK cells can regulate cell function(s) via a mechanism other than cytotoxicity. In addition, we have shown that the induction of NK cells by B lymphocytes requires close interactions between the two cell types. Further, while only low-density B lymphocytes activated in vivo are effective inducers of NK cells, high-density, resting B cells can be rendered effective by preactivation with either interleukin-4 or anti-mu.     

Increased Lysis of Stem Cells but Not Their Differentiated Cells by Natural Killer Cells; De-Differentiation or Reprogramming Activates NK Cells

The aims of this study are to demonstrate the increased lysis of stem cells but not their differentiated counterparts by the NK cells and to determine whether disturbance in cell differentiation is a cause for increased sensitivity to NK cell mediated cytotoxicity. Increased cytotoxicity and augmented secretion of IFN-γ were both observed when PBMCs or NK cells were co-incubated with primary UCLA oral squamous carcinoma stem cells (UCLA-OSCSCs) when compared to differentiated UCLA oral squamous carcinoma cells (UCLA-OSCCs). In addition, human embryonic stem cells (hESCs) were also lysed greatly by the NK cells. Moreover, NK cells were found to lyse human Mesenchymal Stem Cells (hMSCs), human dental pulp stem cells (hDPSCs) and human induced pluripotent stem cells (hiPSCs) significantly more than their differentiated counterparts or parental lines from which they were derived. It was also found that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 in monocytes significantly augmented NK cell cytotoxicity and secretion of IFN-γ. Taken together, these results suggest that stem cells are significant targets of the NK cell cytotoxicity. However, to support differentiation of a subset of tumor or healthy untransformed primary stem cells, NK cells may be required to lyse a number of stem cells and/or those which are either defective or incapable of full differentiation in order to lose their cytotoxic function and gain the ability to secrete cytokines (split anergy). Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation for specific elimination of cancer stem cells.     

Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation

The suppressive effect of human natural killer (NK) cells on B cell differentiation induced by pokeweed mitogen (PWM) was investigated. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into low and high density fractions for which NK cells (Large granular lymphocytes, LGL) and T cells were enriched, respectively. These fractionated mononuclear cells were co-cultured with purified autologous B cells in the presence of PWM, and were examined for their helper and suppressor activities on differentiation of B cells to immunoglobulin-(IgM and IgG) producing cells by a highly sensitive reversed hemolytic plaque assay. The T cell-enriched high density fractions provided help for B cell differentiation to levels higher than that of unfractionated mononuclear cells. On the other hand, the NK-enriched low density fractions did not show helper activity, and when added to the culture of B cells plus helper T cells, they markedly suppressed B cell differentiation. This suppressive activity, as well as the NK cytotoxicity of the NK-enriched fractions, was abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1), but not against T cells (OKT3) in the presence of complement. NK cells also suppressed PWM-driven B cell differentiation in the presence of T4+ (helper/inducer T) but not T8+ (cytotoxic/suppressor T) cells; however, they showed no inhibition of soluble factor-induced B cell differentiation assayed in the absence of helper T cells. It is thus concluded that human peripheral blood NK cells exhibit an ability to suppress PWM-driven B cell differentiation, possibly by acting through the effect on helper T cells but not directly on B cells.     

The direct effects of Toll-like receptor ligands on human NK cell cytokine production and cytotoxicity

Toll-like receptor (TLR) ligands are potent inducers of the innate immune system, of which NK and NKT cells play an important role. We examined the direct activation of highly purified human NK and/or NKT cells with known TLR ligands. NK/NKT cells were positive for all known TLR mRNA (TLR1-10). Ligands for TLR2-5 induced production of significant amounts of IFN-gamma by purified NK cells. However, a TLR9 ligand failed to induce significant levels of the cytokine. NK cells were depleted from PBMCs to confirm that they were the main source of IFN-gamma following treatment with TLR ligands, which resulted in a significant decrease in cytokines. The direct effects of TLR ligands on NK cytotoxicity were determined using 51Cr-labeled K562 target cells. Ligands for TLR2-5 were potent inducers of NK cell cytotoxicity, a TLR9 ligand was not. Our results suggest that TLR ligands can directly stimulate and enhance NK cell cytokine production and induce cytotoxic activities.     

Natural killer activity in cloned cytotoxic T lymphocytes: regulation by interleukin 2, interferon, and specific antigen

It has previously been shown that monoclonal antigen-specific mouse CTL lines can be induced to express cytolytic activity with the same specificity as that of splenic natural killer (NK) cells following culture in high concentrations of concanavalin A-induced spleen cell supernatants. In the present experiments, we made use of this in vitro system to explore the regulation of NK activity at the clonal level. Interferon-alpha and interferon-beta and interleukin 2 (IL 2) were potent inducers of NK activity in CTL, demonstrating that these substances can activate NK functions directly without the participation of other cell types. By comparison, IFN-gamma was a poor activator of NK activity in CTL (and also in fresh spleen cells). Three major differences between induction of NK activity by IFN-alpha,beta and IL 2 were noted: IFN induced NK activity selectively without affecting specific cytolysis, whereas IL 2 also enhanced specific killing; IFN acted much more rapidly than IL 2; and IFN did not induce the cells to enter the cell cycle nor were there any obvious morphologic changes. Specific antigen was also a strong inducer of NK activity in CTL, but studies with antisera against the various classes of IFN revealed that this effect was mediated, at least in part, via the release of IFN-beta. By contrast, the same antisera had no effect on NK induction by crude TCGF or by highly purified IL 2, indicating that the regulation of NK activity by IL 2 occurs at the clonal level in an IFN-independent manner. Although, IL 2, IFN, and Ag could apparently act alone to induce NK activity, much greater (synergistic) induction was obtained by various combinations of these regulators, suggesting that the delivery of two (or more) signals to the responder cell was required for full expression of the NK state. As with fresh splenic NK cells, the induced NK state in cloned CTL was intrinsically labile as revealed by its rapid decay in the absence of inducers, but it could nonetheless be maintained indefinitely at very high levels in the continued presence of inducers. This clonal system thus displays a responsiveness to regulatory signals exactly analogous to that of splenic NK cells and provides a unique and exciting opportunity to evaluate the biochemistry of the regulation of NK activity.     

Loss of NK stimulatory capacity by plasmacytoid and monocyte-derived DC but not myeloid DC in HIV-1 infected patients

Dendritic cells (DC) are potent inducers of natural killer (NK) cells. There are two distinct populations in blood, myeloid (mDC) and plasmacytoid (pDC) but they can also be generated In vitro from monocytes (mdDC). Although it is established that blood DC are lost in HIV-1 infection, the full impact of HIV-1 infection on DC-NK cell interactions remains elusive. We thus investigated the ability of pDC, mDC, and mdDC from viremic and anti-retroviral therapy-treated aviremic HIV-1+ patients to stimulate various NK cell functions. Stimulated pDC and mdDC from HIV-1+ patients showed reduced secretion of IFN-α and IL-12p70 respectively and their capacity to stimulate expression of CD25 and CD69, and IFN-γ secretion in NK cells was also reduced. pDC activation of NK cell degranulation in response to a tumour cell line was severely reduced in HIV-1+ patients but the ability of mDC to activate NK cells was not affected by HIV-1 infection, with the exception of HLA-DR induction. No differences were observed between viremic and aviremic patients indicating that anti-retroviral therapy had minimal effect on restoration on pDC and mdDC-mediated activation of NK cells. Results from this study provide further insight into HIV-1 mediated suppression of innate immune functions.     

Strategies to Rescue Mesenchymal Stem Cells (MSCs) and Dental Pulp Stem Cells (DPSCs) from NK Cell Mediated Cytotoxicity

Background

The aim of this paper is to study the function of allogeneic and autologous NK cells against Dental Pulp Stem Cells (DPSCs) and Mesenchymal Stem Cells (MSCs) and to determine the function of NK cells in a three way interaction with monocytes and stem cells.

Methodology/Principal Findings

We demonstrate here that freshly isolated untreated or IL-2 treated NK cells are potent inducers of cell death in DPSCs and MSCs, and that anti-CD16 antibody which induces functional split anergy and apoptosis in NK cells inhibits NK cell mediated lysis of DPSCs and MSCs. Monocytes co-cultured with either DPSCs or MSCs decrease lysis of stem cells by untreated or IL-2 treated NK cells. Monocytes also prevent NK cell apoptosis thereby raising the overall survival and function of NK cells, DPSCs or MSCs. Both total population of monocytes and those depleted of CD16⁺ subsets were able to prevent NK cell mediated lysis of MSCs and DPSCs, and to trigger an increased secretion of IFN-γ by IL-2 treated NK cells. Protection of stem cells from NK cell mediated lysis was also seen when monocytes were sorted out from stem cells before they were added to NK cells. However, this effect was not specific to monocytes since the addition of T and B cells to stem cells also protected stem cells from NK cell mediated lysis. NK cells were found to lyse monocytes, as well as T and B cells.

Conclusion/Significance

By increasing the release of IFN-γ and decreasing the cytotoxic function of NK cells monocytes are able to shield stem cells from killing by the NK cells, resulting in an increased protection and differentiation of stem cells. More importantly studies reported in this paper indicate that anti-CD16 antibody can be used to prevent NK cell induced rejection of stem cells.     

Augmentation of mouse natural killer cell activity by interferon and interferon inducers.

Interferon (IF), in addition to its anti-viral capacity, is increasingly being found to be a regulator of cell division, cell surface antigens, and cell function. To determine whether IF also plays a role in the regulation of natural killer (NK) cell activity in mice, the in vivo and in vitro effects of IF and IF inducers on NK activity were studied. We observed that pyran, lipopolysaccharide, and polyinosinicopolycytidylic acid (poly I:C) as well as crude and purified IF preparations significantly elevated splenic NK levels in normal mice within 3 to 24 hr of i.p. administration. Normal spleen cells treated with poly I:C or IF in vitro also had augmented NK activity. Poly I:C and IF were themselves not cytotoxic and their presence was not required during the lytic process, indicating that IF acts on lymphocytes to activate NK function. The addition of anti-IF in the incubation medium completely blocked the boosting of NK activity by poly I:C or IF. The characteristics of the effector cells activated by IF were consistent with those of NK cells rather than macrophages, since the boosted effector cells were not retained by a rayon column or removed by carbonyl iron. Moreover, they were resistant to treatment with anti-Thy 1.2 serum plus complement, which eliminated mature T cells.     

Tumor cell differentiation modulates susceptibility to natural killer cells

Induced differentiation in three human cell lines altered their sensitivity specifically to human natural killer (NK) cells by affecting their expression of NK target antigens. Differentiation of HL-60, a promyelocytic leukemia cell line, and the erythroleukemic cell line K562 was accompanied by a concomitant decrease in susceptibility to NK-mediated lysis whereas induction of MeWo melanoma cells resulted in an enhanced sensitivity to lysis. Our findings suggest that target cell susceptibility to NK-mediated lysis may in part be dependent on the stage of differentiation of the tumor cell target.     

Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized,Double-Blind Study

Enhancing antiviral host defense responses through nutritional supplementation would be an attractive strategy in the fight against influenza. Using inoculation with live attenuated influenza virus (LAIV) as an infection model, we have recently shown that ingestion of sulforaphane-containing broccoli sprout homogenates (BSH) reduces markers of viral load in the nose. To investigate the systemic effects of short-term BSH supplementation in the context of LAIV-inoculation, we examined peripheral blood immune cell populations in non-smoking subjects from this study, with a particular focus on NK cells. We carried out a randomized, double-blinded, placebo-controlled study measuring the effects of BSH (N = 13) or placebo (alfalfa sprout homogenate, ASH; N = 16) on peripheral blood mononuclear cell responses to a standard nasal vaccine dose of LAIV in healthy volunteers. Blood was drawn prior to (day-1) and post (day2, day21) LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated, and assessed for surface markers, intracellular markers, and cytotoxic potential by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21) and T cell (day2) populations. LAIV decreased NK cell CD56 and CD158b expression, while significantly increasing CD16 expression and cytotoxic potential (on day2). BSH supplementation further increased LAIV-induced granzyme B production (day2) in NK cells compared to ASH and in the BSH group granzyme B levels appeared to be negatively associated with influenza RNA levels in nasal lavage fluid cells. We conclude that nasal influenza infection may induce complex changes in peripheral blood NK cell activation, and that BSH increases virus-induced peripheral blood NK cell granzyme B production, an effect that may be important for enhanced antiviral defense responses.Trial Registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01269723","term_id":"NCT01269723"}}NCT01269723     

Proteases stimulate mouse erythroleukemia cell differentiation and multiplication

Seventeen different commercially available proteases stimulate both mouse erythroleukemia (MEL) cell differentiation and multiplication. These are the first enzymes shown to stimulate these processes in Friend leukemia virus-infected MEL cells. The induction of differentiation by proteases can be synergistically enhanced by the addition of low concentrations of dimethyl sylfoxide or other low molecular weight inducers. Since proteases are the first inducers of differentiation with a known biochemical function, their study should facilitate the understanding of the molecular mechanism of this process.     

Dissecting NK cell development using a novel alymphoid mouse model: investigating the role of the c-abl proto-oncogene in murine NK cell differentiation

NK lymphocytes participate in both innate and adaptive immunity by their prompt secretion of cytokines including IFN-gamma, which activates macrophages, and by their ability to lyse virally infected cells and tumor cells without prior sensitization. Although these characteristics of NK cells are well documented, little is known about the genetic program that orchestrates NK development or about the signaling pathways that trigger NK effector functions. By crossing NK-deficient common gamma-chain (gammac) and recombinase activating gene (RAG)-2 mutant mice, we have generated a novel alymphoid (B-, T-, and NK-) mouse strain (RAG2/gammac) suitable for NK complementation in vivo. The role of the c-abl proto-oncogene in murine NK cell differentiation has been addressed in hemopoietic chimeras generated using RAG2/gammac mice reconstituted with c-abl-/- fetal liver cells. The phenotypically mature NK cells that developed in the absence of c-abl were capable of lysing tumor targets, recognizing "missing self," and performing Ab-dependent cellular cytotoxicity. Taken together, these results exclude any essential role for c-abl in murine NK cell differentiation in vivo. The RAG2/gammac model thereby provides a novel approach to establish a genetic map of NK cell development.     

Defective differentiation of natural killer cells in SJL mice. Role of the thymus

As previously shown, three distinct phenotypes exist in murine natural killer (NK) cell activity when it is evaluated by the endogenous levels of activity and the susceptibility to augmentation by interferon (IFN) and IFN inducers. The "low" phenotype has low levels of activity which can be poorly augmented by IFN, as in mice of SJL strain. The "inducible" phenotype exhibits low endogenous levels but can vigorously respond to IFN-mediated augmentation, as in A.SW strain. The "high" NK phenotype shows high levels of endogenous activity which can be augmented to still higher levels by IFN, as in B10.S mice. Since SJL mice with congenital absence of the thymus (nude) were of the inducible type, the effect of neonatal thymectomy was examined in the present study. Neonatal thymectomy was found to convert the low phenotype of SJL mice to the inducible, mimicking the effect of nu/nu genotype. Thymectomy as late as 25 days after birth was effective, but retransplantation of a syngeneic newborn or adult thymus, or thymocytes, failed to reverse the effect of thymectomy. The poor responsiveness of NK activity to IFN in SJL, therefore, is extrinsic to the NK cell lineage and is attributable to suppression or maturational block of NK cell differentiation by the thymus during the first few weeks of neonatal life. A series of experiments with bone marrow chimeras showed that the SJL recipients did not allow the expression of inducible or high phenotype by bone marrow progenitors from allogeneic donors with either phenotype. Therefore, the SJL recipients provide an environment which suppresses not only the development of IFN-sensitive NK cell precursors, but also the levels of endogenous NK cell activity. SJL bone marrow cells gave rise to NK activity of inducible phenotype in B10.S recipients, confirming the crucial role of the environment in which NK cell differentiation takes place.     

The BCR/ABL transgene causes abnormal NK cell differentiation and can be found in circulating NK cells of advanced phase chronic myelogenous leukemia patients.

NK cells from the blood of chronic myelogenous leukemia (CML) patients are progressively decreased in number as the disease progresses from chronic phase to blast crisis. We hypothesize that BCR/ABL may be directly responsible by interfering with NK cell differentiation. CD34(+)HLA-DR(+) cells from CML patients were studied for their capacity to differentiate into NK cells. The NK cell cloning frequency was significantly decreased from CML CD34(+)HLA-DR(+) cells compared with cells from normal donors, yet CD34(+)HLA-DR(+) cells gave rise to BCR/ABL(+) NK cells in some patients. This finding prompted us to further investigate circulating NK cells from the blood of CML patients. CD56(+)CD3(-) NK cells were sorted from CML patients and examined by fluorescence in situ hybridization (FISH). In contrast to chronic phase CML, significant numbers of NK cells from advanced phase CML patients were BCR/ABL(+), whereas T cells were always BCR/ABL(-) regardless of the disease stage. To test the effects of BCR/ABL as the sole genetic abnormality, BCR/ABL was transduced into umbilical cord blood CD34(+) cells, and NK development was studied. p210-enhanced green fluorescence protein-transduced cells gave rise to significantly decreased numbers of NK cells compared with enhanced green fluorescence protein transduction alone. In addition, the extrinsic addition of BCR/ABL-transduced autologous CD34(+) cells suppressed the NK cell differentiation of normal umbilical cord blood CD34(+)CD38(-) cells. This study provides the first evidence that BCR/ABL is responsible for the altered differentiation of NK cells and that the NK cell lineage can be involved with the malignant clone in advanced stage CML.     

Roles for common cytokine receptor gamma-chain-dependent cytokines in the generation, differentiation, and maturation of NK cell precursors and peripheral NK cells in vivo

NK cells differentiate in adult mice from bone marrow hemopoietic progenitors. Cytokines, including those that signal via receptors using the common cytokine receptor gamma-chain (gamma(c)), have been implicated at various stages of NK cell development. We have previously described committed NK cell precursors (NKPs), which have the capacity to generate NK cells, but not B, T, erythroid, or myeloid cells, after in vitro culture or transfer to a fetal thymic microenvironment. NKPs express the CD122 Ag (beta chain of the receptors for IL-2/IL-15), but lack other mature NK markers, including NK1.1, CD49b (DX5), or members of the Ly49 gene family. In this report, we have analyzed the roles for gamma(c)-dependent cytokines in the generation of bone marrow NKP and in their subsequent differentiation to mature NK cells in vivo. Normal numbers of NKPs are found in gamma(c)-deficient mice, suggesting that NK cell commitment is not dependent on IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21. Although IL-2, IL-4, and IL-7 have been reported to influence NK cell differentiation, we find that mice deficient in any or all of these cytokines have normal NK cell numbers, phenotype, and effector functions. In contrast, IL-15 plays a dominant role in early NK cell differentiation by maintaining normal numbers of immature and mature NK cells in the bone marrow and spleen. Surprisingly, the few residual NK cells generated in absence of IL-15 appear relatively mature, expressing a variety of Ly49 receptors and demonstrating lytic and cytokine production capacity.     

Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.     

Tumoricidal potential of native blood dendritic cells: direct tumor cell killing and activation of NK cell-mediated cytotoxicity

Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity for DC-based cancer vaccination protocols. Novel findings reveal that besides their role as potent inducers of tumor-specific T cells, human DCs display additional antitumor effects. Most of these data were obtained with monocyte-derived DCs, whereas studies investigating native blood DCs are limited. In the present study, we analyze the tumoricidal capacity of M-DC8(+) DCs, which represent a major subpopulation of human blood DCs. We demonstrate that IFN-gamma-stimulated M-DC8(+) DCs lyse different tumor cell lines but not normal cells. In addition, we show that tumor cells markedly enhance the production of TNF-alpha by M-DC8(+) DCs via cell-to-cell contact and that this molecule essentially contributes to the killing activity of M-DC8(+) DCs. Furthermore, we illustrate the ability of M-DC8(+) DCs to promote proliferation, IFN-gamma production, and tumor-directed cytotoxicity of NK cells. The M-DC8(+) DC-mediated enhancement of the tumoricidal potential of NK cells is mainly dependent on cell-to-cell contact. These results reveal that, in addition to their crucial role in activating tumor-specific T cells, blood DCs exhibit direct tumor cell killing and enhance the tumoricidal activity of NK cells. These findings point to the pivotal role of DCs in triggering innate and adaptive immune responses against tumors.     

Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

Natural killer (NK) cells are innate immune effector cells that protect against cancer and some viral infections. Until recently, most studies have investigated the molecular signatures of human or mouse NK cells to identify genes that are specifically expressed during NK cell development. However, the mechanism regulating NK cell development remains unclear. Here, we report a regulatory network of potential interactions during in vitro differentiation of human NK cells, identified using genome-wide mRNA and miRNA databases through hierarchical clustering analysis, gene ontology analysis and a miRNA target prediction program. The microRNA (miR)-583, which demonstrated the largest ratio change in mature NK cells, was highly correlated with IL2 receptor gamma (IL2Rγ) expression. The overexpression of miR-583 had an inhibitory effect on NK cell differentiation. In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3′ UTR. Therefore, we show that miR-583 acts as a negative regulator of NK cell differentiation by silencing IL2Rγ. Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.     

Interleukin-4 inhibits the differentiation of mouse myeloid leukemia M1 cells induced by dexamethasone, D-factor/leukemia inhibitory factor and interleukin-6, but not by 1 alpha,25-dihydroxyvitamin D3

The effects of interleukin-4(IL-4) on the growth and differentiation of mouse myeloid leukemia M1 cells induced by various differentiation inducers were investigated. IL-4 alone did not have any significant effect on the growth or differentiation of M1 cells, but inhibited their differentiation induced by dexamethasone, D-factor/leukemia inhibitory factor, or interleukin 6. IL-4 also restored the proliferation of M1 cells after growth inhibition during their induction of differentiation by inducers. In contrast, IL-4 enhanced inhibition of growth and induction of differentiation of M1 cells by 1 alpha,25-dihydroxyvitamin D3. These results indicate that modulation of differentiation of M1 cells by IL-4 depends on the differentiation inducer.