Natural Killer Cell Stimulatory Factor Research Articles

Cloning of IL-12, a Critical Advance in Th1 and Th17 Discovery

This Pillars of Immunology article is a commentary on “Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells,” a pivotal article written by S. F. Wolf, P. A. Temple, M. Kobayashi, D. Young, M. Dicig, L. Lowe, R. Dzialo, L. Fitz, C. Ferenz, R. M. Hewick, et al., and published in The Journal of Immunology, in 1991. https://doi.org/10.4049/jimmunol.146.9.3074.

CATALYTIC RECEPTORS

CATALYTIC RECEPTORS

DENDRITIC CELL BASED VACCINES: PROGRESS IN IMMUNOTHERAPY STUDIES FOR PROSTATE CANCER

No effective treatment is currently available for metastatic prostate cancer. Dendritic cell (DC) based cancer vaccine research has emerged from the laboratories to human clinical trials. We describe progress in the development of DC based prostate cancer vaccine. The literature was reviewed for major contributions to a growing number of studies that demonstrate the potential of DC based immunotherapeutics for prostate cancer. Background topics relating to DC based immunotherapy theory and practice are also addressed. DCs have been recognized as the most efficient antigen presenting cells that have the capacity to initiate naive T cell response in vitro and in vivo. During their differentiation and maturation pathways, dendritic cells can efficiently capture, process and present antigens for T cell activation. These characteristics make DC an attractive choice as the cellular adjuvant for cancer vaccines. Advances in DC generation, loading, and maturation methodologies have made it possible to generate clinical grade vaccines for various human trials. More than 100 DC vaccine trials, including 7 studies of patients with advanced prostate cancer have been reported to date. These vaccines were generally well tolerated with no significant adverse toxicity reported. Clinical responders have been identified in these studies. The new prospects opened by DC based vaccines for prostate cancer are fascinating. When compared to conventional treatments, DC vaccinations have few side effects. Improvements in patient selection, vaccine delivery strategies, immune monitoring and vaccine manufacturing will be crucial in moving DC based prostate cancer vaccines closer to the clinics.

Changes in cytokine expression in peripheral leukocytes, detected by microarray analysis, in the secretory vs proliferative phase of the menstrual cycle

Objective: Many autoimmune disorders have recognized clinical changes in pregnancy and the menstrual cycle. However the mechanisms by which hormones, menstrual cycle changes and pregnancy affect the immune system in unknown. The complexity of the immune system and abundance of immune markers necessitate global analysis. Herein, we describe high density oligonucleotide microarray profiling of peripheral blood leukocytes from the proliferative and secretory phases of the menstrual cycle. Design: Microarray analysis of peripheral blood leukocytes from the proliferative and secretory phases of the menstrual cycle. Materials/Methods: Peripheral blood was obtained from healthy female volunteers (n=5) after informed consent. Subjects were 25–34 yo with menstrual cycles of 28–30 days. Paired samples were collected from subjects in nonconception cycles in the late proliferative (cycle day 10–12) and midsecretory phase (day 20–24). Total RNA was extracted using the Paxgene Blood RNA Validation Kit. cDNA was synthesized from total RNA then biotinylated cRNAs were generated by in vitro transcription. Fragmented cRNAs were hybridized on Affymetrix HU95A microarrays, containing 12,686 human genes and ESTs. The data were analyzed with GeneChip Analysis Suite v5.0, GeneSpring v4.2& MS Excel. Results from each phase were normalized and gene expression was compared. Non-parametric testing was applied to genes showing 1.5 fold changes between the proliferative and secretory phases with a p <0.05 for statistical significance. Results: 10 cc of blood yielded 10–15 g of leukocyte RNA. 17 g of high purity, labeled cRNA was used for hybridization. 314 genes were found to be significantly up-regulated in the secretory phase compared to proliferative, including NKG2F, complement receptor C1r, fibroblast growth factor, monocyte chemotactic protein-2, C-C chemokine receptor-4, platelet derived growth factor receptor and natural killer cell stimulatory factor (IL 12). 362 genes were significantly down-regulated in the secretory phase, such as allograft inflammatory factor-1(AIF-1), pre-B cell enhancing factor (PBEF), GM-CSF receptor beta chain, leukocyte immunoglobulin-like receptor-3(LIR-3), interferon regulatory factor 1, lymphocyte activation antigen CD30, and pT49. Conclusions: We have shown significant changes in the expression of cytokines and their receptors in the menstrual cycle. While validation of these findings and further study are needed to determine their clinical significance, immune cells and their effector cytokines are actively involved in the process of implantation and maternal tolerance to a developing fetus. Peripheral changes in immune cells have been previously described in patients with recurrent spontaneous abortion and unexplained infertility. This study lays foundation for further deliniation of steroid hormone control of immune function and dysfunction in women. Supported by: NIH Training grant HD07493 and Women’s Health at Stanford.

Interleukin-12 augments cytolytic activity of peripheral blood mononuclear cells against autologous lung cancer cells in combination with IL-2

The majority of patients with advanced lung cancer die within a few years. Accordingly, new therapeutic modalities need to be developed. Interleukin (IL)-12 was previously known as natural killer (NK) cell stimulatory factor or cytotoxic lymphocyte maturation factor. By virtue of its effects on T cells and NK cells, IL-12 seems to be one of the key cytokines that regulates cell-mediated anti tumor immune responses. Recently, there has been a substantial interest in the potential applications of IL-12 in the treatment of lung cancer. However, there have been no reports about the effect of IL-12 on peripheral blood mononuclear cells (PBMCs) obtained from lung cancer patients in an autologous setting. In this study, we examined the cytotoxicity of PBMC activated by IL-2, IL-12 or both against K562 or autologous lung cancer cells. In contrast to the effect of IL-2 on NK activity, IL-12 alone augmented NK activity against K562 cells, but not against autologous lung cancer cells. IL-12 augmented the IL-2 mediated cytotoxicity of PBMC against both K562 and autologous lung cancer cells. In the absence of IL-2, IL-12 alone cannot induce an autologous anti-tumor effect in vivo. In summary, our results clearly demonstrated that IL-12 can augment the cytolytic activity of PBMC against K562 and autologous lung cancer cells when combined with IL-2, although, IL-12 alone was unable to induce a marked increase in the cytotoxicity against autologous lung cancer cells. These results suggest that an administration of IL-12 in combination with IL-2 may be a useful therapeutic option for solid tumors.

Effect of brief maximal exercise on circulating levels of interleukin-12.

Interleukin-12 (IL-12) is a cytokine that was originally identified as natural killer cell stimulatory factor. It induces the activity of T-helper 1 (Th1) cells and exhibits strong anti-tumor activity. In this study, we studied the effects of brief anaerobic maximal exercise on circulating levels of IL-12. Six healthy males [mean (SD) 25.2 (2.6) years] performed a modified Wingate test exercise (resistance 0.075 kg/kg of body mass). The exercise consisted of five bouts of maximal cycling for 10 s, with rest intervals of 50 s between them. Blood samples were taken before, immediately after, 30 min after, 60 min after and 120 min after the exercise. Plasma concentrations of IL-12 were measured using an enzyme-linked immunosorbent assay. Data were corrected for hemoglobin and hematocrit measurements. Plasma concentrations of IL-12 averaged [mean (SD)] 234.2 (40.9) pg/ml before, 305.2 (62.1) pg/ml immediately after, 202.8 (24.2) pg/ml 30 min after, 239.7 (35.1) pg/ml 60 min after, and 199.6 (49.2) pg/ml 120 min after the exercise. We showed that plasma concentrations of IL-12 increased significantly immediately after brief anaerobic maximal cycle ergometer exercise (P < 0.01).

Antitumor activity of killer cells stimulated with both interleukin-2 and interleukin-12 on mouse glioma cells.

Interleukin-12 (IL-12), originally called natural killer cell stimulatory factor or cytotoxic lymphocyte maturation factor, has potential for use as an immunomodulator in cancer therapy because it significantly retards the growth of some murine tumors. In this study, we analyzed the antitumor effects of lymphocytes stimulated in vitro with both recombinant IL-2 (rIL-2) and rIL-12. When IL-12 was added to mouse splenocytes (SPCs) or human peripheral blood monocytes (PBMCs) incubated with IL-2 for > 4 days, IL-2-induced cytotoxicity against glioma cells was augmented. In contrast, IL-12 inhibited IL-2-induced lymphokine-activated killer (LAK) cell activity when added concurrently to cultures. The concentration of IL-10 induced by IL-12 increased in the supernatant of human PBMCs costimulated with IL-2 and IL-12. Endogenous IL-10 augmented the cytotoxicity of SPCs stimulated with IL-2 or IL-12 or both. However, tumor-bearing mice treated with PBMCs stimulated with both IL-2 and IL-12 did not survive longer than those treated with PBMCs stimulated with IL-2 alone (LAK cells).

IL-12 indirectly enhances proliferation of 5-FU-treated human hematopoietic peripheral blood CD34+ cells.

Interleukin-12 (IL-12) or natural killer cell stimulatory factor (NKSF), has multiple effects on T lymphocytes and natural killer cells. In this study, the effect of IL-12 on human hematopoiesis was studied by analyzing the growth of CD34+ peripheral blood stem cells (PBSC), in steady state. In the presence of Epo, IL-12 alone or in combination with IL-3 or SCF had no effect on the formation of colonies from CD34+ cells. In culture with Epo, G-CSF, and IL-3, the effect of Flt3-ligand (FL) on CD34+ PBSC was investigated in the presence or absence of IL-12. No additional effect of IL-12 was observed when combined with FL. We evaluated 5-FU-treated human CD34+ PBSC proliferation in cultures with Epo, G-CSF, and IL-3, in the presence or absence of IL-12. No cytokine combination enhanced colony formation from 5-FU-treated CD34+ cells. However, in cultures of 5-FU-treated human CD34+ cells, the most efficient combination was IL-3 + Epo + G-CSF + Accessory cells (CD34-). Furthermore, IL-12 enhanced this colony formation significantly. To investigate whether immature CD34+ cells were responsible for FL or SCF, 5-FU-treated human CD34+ cells were cultured with or without IL-12. Whereas no synergistic effect was observed in combination with IL-12, SCF alone significantly enhanced colony formation. However, the colony number was found to be smaller than with the potent combination of accessory cells in the presence of IL-12. These results indicate that accessory cells, lost in CD34+ cell purification, could be partly responsible for an IL-12 effect on immature human PBSC proliferation.

Quantitation of human IL-12 heterodimer and p40

Interleukin 12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor, is a cytokine secreted by a human B lymphoblastoid (NC-37) cell line when induced in culture with phorbol ester and calcium ionophore. This factor has been purified to homogeneity and shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells. In addition, purified IL-12 stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. The protein is a heterodimer composed of a 40- and a 35-kDa subunit. Amino acid sequence analysis confirmed predicted sequences from the cloned cDNAs of each subunit. Chemical and enzymatic deglycosylation of the heterodimer demonstrated that the 40- and 35-kDa subunits contain 10 and 20% carbohydrate, respectively. Structural analysis of IL-12 using site-specific chemical modification revealed that intact disulfide bonds are essential for bioactivity. The 40-kDa subunit of IL-12 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by immunoblotting as being present in NC-37 cell supernatant solutions in relatively large amounts uncomplexed to the 35-kDa subunit. Previously it had been shown that the 40-kDa subunit alone does not cause the proliferation of activated human T lymphocytes or enhance the cytolytic activity of human natural killer cells. However, results obtained by site-specific chemical modification suggesting that a tryptophan residue is at or near the active site of IL-12 may imply a direct role of the subunit in interacting with the IL-12 receptor. These data may support the recent proposal (D. P. Gearing and D. Cosman (1991)Cell 66, 9–10) that IL-12 consists of a complex of cytokine and soluble receptor.

Antitumor and antimetastatic activity of interleukin-12.

Interleukin-12 (IL-12) was identified independently by two groups. The first group, at Hoffmann-La Roche, was studying a cytokine, named cytotoxic lymphocyte maturation factor (CLMF), that activated cytolytic lymphocyte activities (Stern et al. 1990). The second group, at the Wistar Institute, was evaluating a cytokine, originally named natural killer (NK) cell stimulatory factor (NKSF), that could stimulate NK cell functions (Kobayashi et al. 1989). Once CLMF and NKSF were characterized and cloned, it became clear that this was in fact one molecule, which is now called IL-12. IL-12 has profound effects on both NK and T cells, including the ability to stimulate proliferation, cytolytic activity, and cytokine induction. Furthermore, it plays a major role in regulating the induction of T cell subsets and thus is an important component of the immune response to foreign antigens. Based on these properties, IL-12 was evaluated in animal models of malignancies and shown to have potent antitumor and antimetastatic activities. In this chapter, we will summarize some of the biological effects of IL-12 and show how these relate to the in vivo effects of this cytokine on malignancies. Additional information on IL-12 can be found in a number of review articles (Brunda 1994; Gately et al. 1994a; Brunda and Gately 1994; Trinchieri 1994; Gately and Brunda 1995; Brunda and Gately 1995; Hendrzak and Brunda 1995).

Interleukin-12: a pivotal regulator of cell-mediated immunity.

Interleukin-12 (IL-12) is heterodimeric cytokine which is produced by antigen-presenting cells and serves as a pivotal regulator of T- and NK-cell function. IL-12 was identified independently by investigators at the Wistar Institute [1] and at Hoffmann-La Roche [2], who originally called it natural killer cell stimulatory factor (NKSF) [1] or cytotoxic lymphocyte maturation factor (CLMF) [2], respectively. Cloning of the cDNAs encoding the proteins mediating these 2 activities showed that NKSF and CLMF were the same molecule [3,4], which is now generally referred to as IL-12. The biologic activities initially associated with IL-12 include its ability to enhance the lytic activity of natural killer/lymphokine-activated killer (NK/LAK) cells [1,2], to induce the secretion of interferon-γ (IFN-γ) by both resting and activated T and NK cells [1,5], to stimulate the proliferation of activated T and NK cells [2,6], and to facilitate cytolytic T-lymphocyte (CTL) responses [7,8]. All of these activities are shared by IL-12 with other interleukins such as IL-2. However, IL-12 has more recently been found to play a critical and unique role in promoting TH1-type cytokine responses [9,10], thereby facilitating cell-mediated immunity. The immunoenhancing effects of IL-12 on T and NK cell function provide the basis for its therapeutic effects in a number of mouse tumor models and models of infectious diseases.KeywordsNatural Killer ActivitySoluble Leishmania AntigenRenca TumorStaphylococcus Aureus CowanNatural Killer Cell Stimulatory FactorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Synergism of interleukin 12, interleukin 3 and serum factor on primitive human hematopoietic progenitor cells.

Interleukin 12 (IL-12), or natural killer cell stimulatory factor (NKSF), has multiple biologic effects on T lymphocytes and natural killer cells. In this study, we evaluated the effect of IL-12 on human hematopoiesis by analyzing the growth of CD34+33- cells in methylcellulose culture with or without serum. Blood cells were collected by apheresis from an expanded stem cell pool during the rebound phase of hematopoiesis after chemotherapy and purified by combined purification procedures to yield target CD34+33- cells. IL-12, by itself or in combination with IL-3 and erythropoietin (Epo), had no effect on the formation of colonies from the unfractionated blood mononuclear cells. In the presence of fetal bovine serum (FBS), IL-12 increased the number of colony forming units for granulocyte-macrophage (CFU-GM) from CD34+33- cells which responded to IL-3, but not the number of CFU-GM from those which responded to stem cell factor (SCF) or granulocyte colony stimulating factor (G-CSF). This effect was not abolished by the addition of monoclonal antibodies against tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-1 alpha, IL-6, G-CSF, or granulocyte/macrophage CSF (GM-CSF). However, the stimulatory effect of IL-12 was abolished in secondary plating in serum-containing methylcellulose culture after short-term (48 h) liquid incubation with IL-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Alveolar Macrophages Augment Natural Killer Cell Stimulatory Factor (Interleukin‐12)‐inducible Killer Activity from Autologous Blood Lymphocytes

Interleukin‐12 (IL‐12), also known as natural killer cell stimulatory factor (NKSF), was found to induce cytotoxic activity from human blood T cells and NK cells. The present study was undertaken to examine the effect of human alveolar macrophages (AM) on induction by IL‐12 cytotoxic cells from blood lymphocytes. AM were obtained by bronchoalveolar lavage from healthy donors. Highly purified lymphocytes (>99%) and monocytes (>90%) were also isolated by centrifugal elutriation from peripheral blood of the same donors. Cytotoxicity of lymphocytes was measured by 4‐h 51Cr release assay. IL‐12 stimulated blood lymphocytes to produce interferon γ (IFNγ) and tumor necrosis factor α (TNFα), and this effect was augmented by co‐cultivation with monocytes or AM. AM‐upregulated induction of cytotoxic lymphocytes was stimulated with IL‐12, and this effect was significantly abrogated by addition of antibodies against IFNγ and TNFα. Induction by IL‐12 of IFNγ production and cytotoxic activity of CD8+ cells was also augmented by co‐cultivation with monocytes or AM. AM were more effective than monocytes in augmenting the cytotoxic activity of IL‐12‐stimulated lymphocytes and CD8+ cells. These observations suggest that in situ induction of IL‐12‐stimulated cytotoxic cells in the lung may be regulated by complex cytokine networks, depending on participation of monocytes and alveolar macrophages.

Interleukin-12.

Interleukin (IL)-12 was originally identified as a factor produced by human Epstein-Barr virus-transformed B cell lines. It was detected by one group as cytotoxic lymphocyte maturation factor, a cytokine that synergized with IL-2 in the induction of lymphokine-activated killer cells and cytotoxic T lymphocytes. A second group characterized it as a natural killer (NK) cell stimulatory factor, due to the enhancement of cytotoxicity and IFN-gamma synthesis by NK cells. Human IL-12 was purified to homogeneity and cloned by both groups. We had identified a murine factor, provisionally termed T cell-stimulating factor (TSF), which was involved in the proliferation, synthesis of IFN-gamma and cell adhesion of CD4+ Th1 cells. TSF was produced in the antigen-specific interaction between Th1 cells and macrophages as antigen-presenting cells, partially purified from supernatants of such cultures, and shown to be identical to IL-12. Monocytes/macrophages appear to be the major source of IL-12. It is rapidly produced by phagocytic cells after stimulation with several bacteria/bacterial products and other microorganisms. In the light of its effects on NK cells as well as CD4+ and CD8+ T cells, IL-12 can be regarded as a cytokine that connects the innate immune system with the acquired immunity. IL-12 has a broad range of activities already reviewed in three papers. These include the regulation of cytokine synthesis and proliferation of T and NK cells, the promotion of Th1 cell development, the differentiation of CD8+ T cells and effects on hematopoiesis. When applied in vivo, IL-12 was shown to enhance the resistance to bacterial and parasitic infections, to promote antitumor immunity, and to influence antiviral responses including HIV in vivo or in vitro. This review will briefly summarize these effects, but mainly focus on recent results concerning the regulation of the production and the activity of IL-12, its role in the differentiation of Th cells and the implications for delayed- and immediate-type hypersensitivity reactions, its importance for organ-specific autoimmune diseases, and the possible role of the IL-12p40 homodimer as a specific inhibitor of the IL-12 heterodimer.

Induction mechanism of human blood CD8+ T cell proliferation and cytotoxicity by natural killer cell stimulatory factor (interleukin-12).

Natural killer cell stimulatory factor (NKSFJ IL‐12) has been found to induce cytotoxic activity of human blood T cells. In the present study, the effect of NKSF on induction of cytotoxic CD8+ T cells in the presence or absence of monocytes was examined. Highly purified lymphocytes (>99%) and monocytes (>90%) were isolated hy centrifugal elutriation from peripheral blood of normal donors. Then, CD8+ cells were isolated with antibody‐bound magnetic beads from purified lymphocytes. The cytotoxicity of CD8+ cells was measured by 51Cr release assay for 4 h. NKSF enhanced the proliferative response of CD8+ cells stimulated with suboptimal concentrations of interleukin‐2 (IL‐2), but rather inhibited their proliferative and cytotoxic responses on stimulation with an optimal concentration of IL‐2. NKSF stimulated CD8+ cells to produce interferon 7 (IFNγ) irrespective of the presence of added IL‐2, and this effect was augmented by co‐cultivation with monocytes. Blood monocytes upregulated induction of cytotoxic CD8+ cells stimulated with NKSF alone, and this effect was abolished by addition of antibody against IFNγ, but not of antibody against tumor necrosis factor a. Induction of NKSF‐inducible cytotoxic CD8+ cells was inhibited by addition of transforming growth factor β, but not of IL‐4. These observations suggest that in situ induction of NKSF‐stimulated cytotoxic CD8+ cells may be regulated by complex cytokine networks, depending on the participation of monocytes.

Natural killer cell stimulatory factor (NKSF)/IL-12 and cytolytic activities of PBL/NK cells from human immunodeficiency virus type-1 infected patients.

The aim of this study was to assess the effect of natural killer stimulatory factor (NKSF)/IL-12) on the cytolytic potential of natural killer (NK) cells from normal donors or HIV-1 infected individuals, at different stages of disease. The results obtained confirm the powerful ability of IL-12 to enhance PBLs' cytotoxic activity to tumour cells in normals and in those HIV-1 infected patients, with maintained cytotoxic activity. IL-12 was however poorly effective in enhancing the cytolytic potential of any donors. Similar results were obtained adding IL-2, even at doses as high as 100 IU/ml in patients with CD4 number below 200/mm3. These results should be taken into account when designing therapeutic trials of HIV-1 infection based on cytokines.

Differential regulation of interleukin-12 (IL-12), tumor necrosis factor alpha, and IL-1 beta production in human myeloid leukemia cell lines and peripheral blood mononuclear cells

Natural killer cell-stimulatory factor or interleukin-12 (NKSF/IL-12) was originally identified and purified from the conditioned medium of Epstein-Barr virus (EBV)-transformed B-cell lines. Phorbol diesters were observed to be potent stimulators of NKSF/IL-12 production from the B-cell lines. Although monocytes were found to be the major producers of NKSF/IL-12 in peripheral blood (PB) in response to lipopolysaccharide (LPS) or to Staphylococcus aureus, several myeloid leukemia cell lines tested did not produce detectable NKSF/IL-12 either constitutively or upon stimulation with phorbol diesters. However, three lines, ML-3, HL-60, and THP-1, responded to LPS with significant levels of NKSF/IL-12 production, whereas S aureus was effective only on THP-1 cells. When the cell lines were preincubated with compounds known to induce them to differentiate, production of tumor necrosis factor alpha (TNF alpha) and IL-1 beta was in most cases maximal in cells with differentiated characteristics, whereas NKSF/IL-12 production in response to LPS in all three producing cell lines was significantly enhanced only by pretreatment with dimethylsulfoxide (DMSO) for 24 hours, or by costimulation with interferon gamma (IFN gamma). The efficiency of DMSO enhancement of NKSF/IL-12 production decreased after 2 to 5 days of incubation, when the cells acquired differentiated characteristics. Unlike DMSO, IFN gamma enhanced NKSF/IL-12 production, and IL-10 and dexamethasone inhibited it in cell lines and PB mononuclear cells stimulated by either LPS or S aureus. The ability of the cell lines to respond to these mediators of possibly physiologically relevant function provides a tissue-culture model for studying their mechanism of action.

Cytokine cross-talk between phagocytic cells and lymphocytes: relevance for differentiation/activation of phagocytic cells and regulation of adaptive immunity.

Cytokines represent one of the most important elements in the communication among different cell types. They play an increasingly better understood role in the communication among hematopoietic cells and in particular in the reciprocal regulation of effector cell types of innate or natural resistance (phagocytic cells and Natural Killer (NK) cells) and those of adaptive immunity (T and B lymphocytes). Lymphocytes produce several cytokines with either stimulatory (e.g., colony stimulatory factor) or suppressive (e.g., tumor necrosis factors and interferons) effects on proliferation of early hematopoietic cells. Many of these cytokines, alone or acting in synergistic combinations, also have a differentiation-inducing ability on immature myeloid cells and act as powerful potentiators of the cellular functions of terminally differentiated phagocytic cells. The communication between lymphocytes and phagocytic cells is not unidirectional, as phagocytic cells produce factors that regulate lymphocyte activation. In addition to their role as antigen presenting cells expressing costimulatory accessory molecules and secreting cytokines (e.g., IL-1, IL-6, TNF), phagocytic cells have been recently shown to produce Natural Killer cell Stimulatory Factor (NKSF/IL-12). IL-12 is a heterodimeric cytokine with important modulatory functions on cytotoxicity of NK and T cells, lymphocyte proliferation, lymphokine production, and development of T helper cell subsets. These communications between phagocytic cells and lymphocytes are further regulated by negative and positive feedback mechanisms that contribute to maintain the homeostasis of the system in physiologic conditions and to govern the changes in this equilibrium needed for the response to infectious or other foreign agents.

Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells.

Natural killer cell stimulatory factor or interleukin 12 (NKSF/IL-12) is a heterodimeric cytokine produced by monocytes/macrophages, B cells, and possibly other accessory cell types primarily in response to bacteria or bacterial products. NKSF/IL-12 mediates pleiomorphic biological activity on T and NK cells and, alone or in synergy with other inducers, is a powerful stimulator of interferon gamma (IFN-gamma) production. IL-10 is a potent inhibitor of monocyte-macrophage activation, that inhibits production of tumor necrosis factor alpha (TNF-alpha), IL-1 and also IFN-gamma from lymphocytes acting at the level of accessory cells. Because TNF-alpha and IL-1 are not efficient inducers of IFN-gamma, the mechanism by which IL-10 inhibits IFN-gamma production is not clear. In this paper, we show that IL-10 is a potent inhibitor of NKSF/IL-12 production from human peripheral blood mononuclear cells activated with Staphylococcus aureus or lipopolysaccharide (LPS). Both the production of the free NKSF/IL-12 p40 chain and the biologically active p70 heterodimer are blocked by IL-10. NKSF/IL-12 p40 chain mRNA accumulation is strongly induced by S. aureus or LPS and downregulated by IL-10, whereas the p35 mRNA is constitutively expressed and only minimally regulated by S. aureus, LPS, or IL-10. Although IL-10 is able to block the production of NKSF/IL-12, a powerful inducer of IFN-gamma both in vitro and in vivo, the mechanism of inhibition of IFN-gamma by IL-10 cannot be explained only on the basis of inhibition of NKSF/IL-12 because IL-10 can partially inhibit IFN-gamma production induced by NKSF/IL-12, and also, the IFN-gamma production in response to various stimuli in the presence of neutralizing antibodies to NKSF/IL-12. Our findings that antibodies against NKSF/IL-12, TNF-alpha, or IL-1 beta can significantly inhibit IFN-gamma production in response to various stimuli and that NKSF/IL-12 and IL-1 beta can overcome the IL-10-mediated inhibition of IFN-gamma, suggest that IL-10 inhibition of IFN-gamma production is primarily due to its blocking production from accessory cells of the IFN-gamma-inducer NKSF/IL-12, as well as the costimulating molecule IL-1 beta.

Enhancing effect of natural killer cell stimulatory factor (NKSF/interleukin‐12) on cell‐mediated cytotoxicity against tumor‐derived and virus‐infected cells

Natural killer cell stimulatory factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine with pleiomorphic effects on T and NK cells, including induction of lymphokine production, mitogenesis, and enhancement of spontaneous cytotoxic activity. Similarly to IL-2, NKSF/IL-12 enhances NK cell-mediated cytotoxicity within a few hours and independently from induced proliferation. This effect is independent from other induced cytokines, because it is not prevented by antibodies neutralizing interferon (IFN)-alpha, IFN-beta, IFN-gamma, IL-2 or tumor necrosis factor (TNF)-alpha and, unlike the induction of IFN-gamma production by peripheral blood lymphocytes, it does not require HLA class II-positive accessory cells. Enhanced cytotoxicity is accompanied by morphologic changes in NK cells, including a significant increase in the number of cytoplasmic granules. In addition to the previously described ability to enhance the cytotoxic activity of NK cells against tumor-derived target cells, NKSF/IL-12 is also a potent stimulator of cytotoxicity against virus-infected cells, either fibroblasts acutely infected with herpes viruses or T cell lines chronically infected with human immunodeficiency virus-1. NK cell-mediated antibody-dependent cytotoxicity or anti-CD16 antibody-redirected lysis is not significantly enhanced by NKSF/IL-12. However, the ability of resting peripheral blood T cells to mediate anti-CD3 antibody-redirected lysis is enhanced by 18-h incubation with NKSF/IL-12, indicating that this lymphokine can modulate the cytotoxic capability of both NK and T cells.