Regulation of human natural killing. II. Protective effect of interferon on NK cells from suppression by PGE2

Activation of human natural killer (NK) cells in vitro with interferon (IFN) and poly I:C results in a partial loss of sensitivity of these cells to suppression by PGE2. The acquired resistance to suppression can be induced with the large granular lymphocytes (LGL) in the absence of monocytes. With K562, HSB, and CEM used as NK target cells, the IFN-induced resistance to suppression by PGE2 is observed with all three target cells. Furthermore, ADCC activity of IFN-activated cells against tumor (SB-TNP) and erythroid (CRC-TNP) target cells is also less susceptible to suppression by PGE2. The dual effect of IFN on NK cells is prompt; the augmentation of NK activity and the acquired resistance to suppression by PGE2 can be seen after 3 hr of treatment with IFN. Both of these characteristics seem to be quite stable for at least 24 hr. Spleen cells from mice (CBA, C3H, and BALB/c nude) treated in vivo with poly I:C also acquire partial resistance to suppression by PGE2. Our data therefore suggest that IFN-stimulated NK cells are protected from suppression by PGE2. Biologically, the IFN-induced protective effect may be beneficial to host resistance to neoplasia.     

IL-10 reduces Th2 cytokine production and eosinophilia but augments airway reactivity in allergic mice

Mononuclear phagocytes can interact with mesenchymal cells and extracellular matrix components that are crucial for connective tissue rearrangement. We asked whether blood monocytes can alter matrix remodeling mediated by human lung fibroblasts cultured in a three-dimensional collagen gel. Blood monocytes from healthy donors (>95% pure) were cast into type I collagen gels that contained lung fibroblasts. Monocytes in coculture inhibited the fibroblast-mediated gel contractility in a time- and concentration-dependent manner. The concentration of PGE(2), a well-known inhibitor of gel contraction, was higher (P < 0.01) in media from coculture; this media attenuated fibroblast gel contraction, whereas conditioned media from either cell type cultured alone did not. Three-dimensional cultured monocytes responded to conditioned media from cocultures by producing interleukin-1beta and tumor necrosis factor-alpha, whereas fibroblasts increased synthesis of PGE(2). Antibodies to interleukin-1beta and tumor necrosis factor-alpha blocked the monocyte inhibitory effect and reduced the amount of PGE(2) produced. The ability of monocytes to block the fibroblast contraction of matrix may be an important mechanism in regulating tissue remodeling.     

A short synthetic peptide fragment of human interleukin-1 beta increases both human and murine natural killer activity

The effect of a short synthetic fragment of human interleukin-1 beta (hu IL-1 beta) on natural killer (NK) activity was examined. Peripheral-blood mononuclear cells (PBMC) from normal donors showed a significant increase in NK activity against K562 leukemia cells after preincubation for 18 h with the IL-1 peptide. A similar augmentation was not observed after culturing the cells in the presence of hu IL-1 beta. The increase in tumor cell lysis could not be ascribed to a cytolytic activity of the synthetic fragment on target cells, since the peptide caused no direct lysis of various tumor cell lines. Although the peptide enhanced NK cytotoxicity of PBMC, highly purified large granular lymphocytes were not susceptible to its stimulatory effect. The addition to the cultures of antibodies to human interleukin-2 (hu IL-2) completely blocked the peptide-induced boost of NK cytotoxicity, suggesting that IL-2 is mainly involved in the activation process. The ability of the IL-1 peptide to increase NK activity was further confirmed in vivo in the mouse. Cytotoxicity against YAC-1 lymphoma cells, which was very low in the spleen of untreated BALB/c mice, was in fact significantly increased after a single inoculation of the peptide. These data thus indicate that a short synthetic peptide fragment of hu IL-1 beta is able to increase both human and murine NK activity.     

The NKp46 receptor contributes to NK cell lysis of mononuclear phagocytes infected with an intracellular bacterium

We used human tuberculosis as a model to investigate the role of NK cytotoxic mechanisms in the immune response to intracellular infection. Freshly isolated NK cells and NK cell lines from healthy donors lysed Mycobacterium tuberculosis-infected monocytes to a greater extent than uninfected monocytes. Lysis of infected monocytes was associated with increased expression of mRNA for the NKp46 receptor, but not the NKp44 receptor. Antisera to NKp46 markedly inhibited lysis of infected monocytes. NK cell-mediated lysis was not due to reduced expression of MHC class I molecules on the surface of infected monocytes or to enhanced production of IL-18 or IFN-gamma. NK cell lytic activity against M. tuberculosis-infected monocytes and NKp46 mRNA expression were reduced in tuberculosis patients with ineffective immunity to M. tuberculosis compared with findings in healthy donors. These observations suggest that 1) the NKp46 receptor participates in NK cell-mediated lysis of cells infected with an intracellular pathogen, and 2) the reduced functional capacity of NK cells is associated with severe manifestations of infectious disease.     

Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes.

Activation of human monocytes results in the production of interstitial collagenase through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Inasmuch as interleukin 4 (IL-4) has been shown to inhibit PGE2 synthesis by monocytes, we examined the effect of IL-4 on the production of human monocyte interstitial collagenase. Additionally, we also assessed the effect of IL-4 on the production of 92-kDa type IV collagenase/gelatinase and tissue inhibitor of metalloproteinase-1 (TIMP-1) by monocytes. The inhibition of PGE2 synthesis by IL-4 resulted in decreased interstitial collagenase protein and activity that could be restored by exogenous PGE2 or dibutyryl cyclic AMP (Bt2cAMP). IL-4 also suppressed ConA-stimulated 92-kDa type IV collagenase/gelatinase protein and zymogram enzyme activity that could be reversed by exogenous PGE2 or Bt2cAMP. Moreover, indomethacin suppressed the ConA-induced production of 92-kDa type IV collagenase/gelatinase. These data demonstrate that, like monocyte interstitial collagenase, the conA-inducible monocyte 92-kDa type IV collagenase/gelatinase is regulated through a PGE2-mediated cAMP-dependent pathway. In contrast to ConA stimulation, unstimulated monocytes released low levels of 92-kDa type IV collagenase/gelatinase that were not affected by IL-4, PGE2, or Bt2cAMP, indicating that basal production of this enzyme is PGE2-cAMP independent. IL-4 inhibition of both collagenases was not a result of increased TIMP expression since Western analysis of 28.5-kDa TIMP-1 revealed that IL-4 did not alter the increased TIMP-1 protein in response to ConA. These data indicate that IL-4 may function in natural host regulation of connective tissue damage by monocytes.     

Monocytes from Depressed Patients Display an Altered Pattern of Response to Endotoxin Challenge

It is now well established that major depression is accompanied and characterized by altered responses of the immune-inflammatory system. In this study we investigated the pro-inflammatory activation of monocytes isolated from depressed patients as a parameter not influenced by such confounds as the time of day, the nutritional and exercise status or the age and gender of patients. Monocytes from depressed patients and from healthy controls were isolated in vitro; after 24-h incubation under basal conditions, cells were exposed for 24-h to 100 ng/ml of endotoxin (bacterial lipopolysaccharide, LPS). We found that monocytes from drug-free depressed patients and controls release the same amounts of prostaglandin E2 (PGE2) under basal conditions, whereas monocytes from patients are dramatically less reactive to LPS (8.62-fold increase vs previous 24 hrs) compared to healthy controls (123.3-fold increase vs previous 24 hrs). Such blunted prostanoid production was paralleled by a reduction in COX-2 gene expression, whereas other pro-inflammatory mediators, namely interleukin-1β (IL-1 β) and -6 (IL-6) showed a trend to increased gene expression. The above changes were not associated to increased levels of circulating glucocorticoids. After 8 months of antidepressive drug treatment, the increase in PGE2 production after the endotoxin challenge was partially restored, whereas the increase in IL-1 β and -6 levels observed at baseline was completely abolished. In conclusion, our findings show that the reactivity of monocytes from depressed patients might be considered as a marker of the immune-inflammatory disorders associated to depression, although the lack of paired healthy controls at follow-up does not allow to conclude that monocyte reactivity to endotoxin is also a marker of treatment outcome.     

Innate immune response of the human host to exposure with herpes simplex virus type 1: in vitro control of the virus infection by enhanced natural killer activity via interleukin-15 induction

Infections with herpes simplex virus type 1 (HSV-1) in humans and in animal models are accompanied by enhanced natural killer (NK) activity. In vitro, HSV-1 also enhances the NK activity of human peripheral blood mononuclear cells (PBMC). The molecular basis of this enhanced NK activity, however, is not well characterized. We investigated the role of human interleukin-15 (IL-15) in this phenomenon and report here that HSV-1-mediated enhanced NK activity was abrogated by neutralizing antibodies for IL-15 but not for other cytokines (i.e., IL-2, IL-12, gamma interferon [IFN-gamma], tumor necrosis factor alpha, or IFN-alpha). Anti-CD122 antibodies which block signaling through IL-2 receptor beta chain, and therefore neutralize the effects of IL-15 (and IL-2), also abrogated this enhancement. Furthermore, HSV-1 increased the levels of IL-15 mRNA and the production of IL-15 in HSV-1-infected PBMC cultures. The neutralization of IL-15 in cocultures of PBMC with HSV-1-infected cells significantly increased HSV-1 production. These results strongly suggest a role for IL-15 in the HSV-1-mediated in vitro enhancement of NK activity and in the PBMC-mediated suppression of HSV-1 replication.     

Immunoregulatory role of in vitro differentiated macrophages on human natural killer (NK)-cell activity

Immunoregulatory effects of human macrophages on natural killer (NK) activity were studied. Monocytes were isolated by adherence to plastic, after leukapheresis of normal blood donors, and cultured for 1 to 14 days. In vitro-differentiated (5-7 days) human macrophages consistently and significantly (P less than 0.01) augmented NK activity of fresh autologous or allogeneic PBMNC. During culture, these macrophages also developed increased antitumor cytostatic activity. The optimal time for both the expression of cytostatic activity and up-regulation of NK activity was 5-7 days in culture. In contrast, 12- to 14-day macrophages significantly suppressed NK activity and had less cytostatic activity. Macrophages in culture demonstrated shifts in Leu-M3+HLA-DR+ phenotype from the mean of 60% +/- 11 (SD) in fresh monocytes to 90% +/- 5 between Days 5 and 7 in culture and then down to 10% +/- 5 in 14-day cultures. The activity of NK (CD56+CD3-) cells, purified by Percoll gradient centrifugation and flow cytometry, was up-regulated directly by in vitro-differentiated macrophages at low macrophage to NK cell ratios, and this up-regulation was not dependent on T lymphocytes or other accessory cells. The modulation of NK activity by differentiated macrophages was not MHC-restricted and depended on the viability and cellular integrity of macrophages. Sonicated macrophages could no longer up-regulate NK activity. This study shows that antitumor effects mediated by human in vitro differentiated LeuM3+HLA-DR+ macrophages may simultaneously involve more than one mechanism, namely direct cytostasis of tumor cells and activation of NK cells.     

Inhibition of human NK cell and LAK cell cytotoxicity and differentiation by PGE2

Activation of natural killer (NK) activity K562 target cells from nonadherent (NA) lymphocytes by interleukin 2 (IL-2) was inhibited marginally PGE2 (30-3000 nM). PGE2 did not effectively suppress the NK activity of IL-2-activated cells. The NK activation and acquisition of resistance to PGE2-mediated suppression of NK activity were dependent on protein synthesis. When NA cells were incubated with IL-2 for 3 or more days to generate lymphokine-activated killer (LAK) activity against Raji target cells, PGE2 only partially inhibited the activation of NK/LAK activity by an optimal dose of IL-2 (10 U/ml). The activation of NK/LAK activity by a suboptimal dose of IL-2 (0.1 U/ml) was inhibited by PGE2. When the NK/LAK activity of IL-2-activated cells was assessed in the presence or absence of PGE2, the LAK activity was more sensitive than the NK activity to PGE2-mediated suppression.     

Synergistic activation of human monocytes by granulocyte-macrophage colony-stimulating factor and IFN-gamma. Increased TNF-alpha but not IL-1 activity

TNF-alpha and IL-1 activities and PGE2 levels were investigated in the supernatants of highly purified human monocytes cultured for 18 h with recombinant human granulocyte-macrophage CSF (GM-CSF). GM-CSF alone did not stimulate IL-1 or TNF-alpha activities or the production of PGE2. GM-CSF with IFN-gamma, but not with LPS, consistently activated the monocytes for TNF-alpha activity. In contrast, for increased IL-1 activity, GM-CSF synergized weakly and irregularly with LPS, but not at all with IFN-gamma. For the third monocyte product investigated, GM-CSF was a weak and inconsistent inducer of PGE2 and only in the co-presence of IFN-gamma. Thus, GM-CSF can elicit different responses in human monocytes depending both on the co-stimulus as well as the monocyte product being investigated.     

Lipopolysaccharide exposure during purification of human monocytes by adherence increases their recovery and cytolytic activity

Exposure of monocytes to lipopolysaccharide (LPS) during, but not after, adherence purification increased their cytolytic activity in short-term 51Cr-release assays against K562 target cells. In the absence of LPS only a minority of monocytes could be recovered by adherence. With 1 ng/ml to 10 micrograms/ml LPS present during the 1-hr adherence procedure, however, monocytes spread more extensively on serum-coated plastic and glass surfaces and virtually all of the monocytes in a mononuclear leukocyte preparation were recovered in the adherent fraction. While increasing the recovery of monocytes threefold, LPS exposure during adherence also increased monocyte purity as assessed by peroxidase staining, morphology, and indirect immunofluorescence with monoclonal Mo2. The proportion of Leu-11-positive NK cells in the adherent fraction did not change. Depletion of NK cells by treatment with anti-Leu-11b and complement eliminated cytolytic activity from the nonadherent, but not from the adherent, fraction isolated with LPS. Thus, addition of LPS during adherence produced a monocyte preparation with enhanced cytolytic activity not attributable to NK contaminants. To test whether LPS caused production of lymphokines that activate monocytes, we tested supernatants of unseparated mononuclear leukocytes for the capacity to stimulate purified monocytes for cytolysis. Such supernatants stimulated monocytes more effectively than LPS alone. We conclude that LPS stimulates monocytes for cytolysis most effectively during adherence purification because LPS allows the recovery of weakly adherent monocytes with high cytolytic capacity; also, LPS may stimulate production of lymphokines that further augment monocyte cytolytic activity.     

D-dimer and CoV-2 spike-immune complexes contribute to the production of PGE2 and proinflammatory cytokines in monocytes

An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8. The DD-induced PGE2 and inflammatory cytokines were enhanced significantly by co-treatment with immune complexes (IC) of SARS CoV-2 recombinant S protein or of pseudovirus containing SARS CoV-2 S protein (PVCoV-2) coated with spike-specific chimeric monoclonal antibody (MAb) containing mouse variable and human Fc regions. The production of PGE2 and cytokines in monocytes activated with DD and ICs was sensitive to the inhibitors of β2 integrin and FcγRIIa, and to the inhibitors of calcium signaling, Mitogen-Activated Protein Kinase (MAPK) pathway, and tyrosine-protein kinase. Importantly, strong increase in PGE2 and in IL-6/IL-8/IL-1β cytokines was observed in monocytes activated with DD in the presence of IC of PVCoV-2 coated with plasma from hospitalized COVID-19 patients but not from healthy donors. The IC of PVCoV-2 with convalescent plasma induced much lower levels of PGE2 and cytokines compared with plasma from hospitalized COVID-19 patients. PGE2 and IL-6/IL-8 cytokines produced in monocytes activated with plasma-containing IC, correlated well with the levels of spike binding antibodies and not with neutralizing antibody titers. Our study suggests that a combination of high levels of DD and high titers of spike-binding antibodies that can form IC with SARS CoV-2 viral particles might accelerate the inflammatory status of lung infiltrating monocytes leading to increased lung pathology in patients with severe form of COVID-19.     

CD40-ligation-mediated protection from apoptosis of a Fas-sensitive Hodgkin's-disease-derived cell line

 Paclitaxel or Taxol has attracted a great deal of attention in recent years because of its immense success as a chemotherapeutic agent for numerous types of cancer. It is known that paclitaxel stabilizes microtubules, and this characteristic is the presumed primary mechanism for its antitumor activity. Recently, however, paclitaxel’s ability to regulate gene expression, particularly in the murine system, has been reported by several groups. Here, we present research examining paclitaxel’s ability to alter expression of the interleukin-1β (IL-1β) and IL-8 cytokines in primary human monocytes, T lymphocytes, and four human breast cancer cell lines: MCF-7, ZR-75-1, MDA-MB-468, and MDA-MB-231. This report shows for the first time that treatment with 5–50 μM paclitaxel increases steady-state levels of IL-1β mRNA in unprimed human monocytes, MCF-7, and ZR-75-1 cells. Monocytes from eight donors in 16 experiments showed increased IL-1β secretion upon treatment; however, the increase in IL-1β production by monocytes was predicated on culturing in the absence of fetal bovine serum or in the presence of autologous human serum. In contrast to the IL-1β results, paclitaxel did not have significant effects on IL-8 expression by monocytes, T lymphocytes, or the breast cancer cells. These data show a specific effect of paclitaxel on cytokine synthesis by both immune cells and cancer cells. Received: 8 September 1997 / Accepted: 26 November 1997     

Brugia malayi Microfilariae Induce a Regulatory Monocyte/Macrophage Phenotype That Suppresses Innate and Adaptive Immune Responses

Background

Monocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive.

Aim

To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses.

Methodology and principal findings

Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4⁺ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10). IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner.

Conclusions and significance

Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic filariasis is caused by microfilaria-modulated monocytes in an IL-10-dependent manner. Together with suppression of macrophage innate responses, this may contribute to the overall down-regulation of immune responses observed in asymptomatically infected patients.     

Immunomodulating effects in vitro of interleukin-2 and interferon-gamma on human blood and bone marrow mononuclear cells

Mononuclear cells (MNC) derived from peripheral blood (PBMNC) of 23 normal donors and 4 AIDS patients, and from bone marrow (BMMNC) of 15 normal donors were incubated at 37 degrees C in culture medium alone or in the presence of either natural or recombinant human interleukin-2 (IL-2) or recombinant human interferon-gamma (IFN-gamma; 1-1,000 U/ml). The cultured cells were washed on days 1, 4 or 7 and tested for various immune functions in vitro and for cell surface phenotype. IL-2, but not IFN-gamma, was found mitogenic for both PBMNC and BMMNC. The natural killer (NK) activity of both PBMNC and BMMNC was the only function tested that was markedly augmented (over 100-fold compared to medium control) by both lymphokines. Pretreatment of PBMNC with IL-2 at greater than or equal to 10 U/ml profoundly suppressed (up to 90%) various functions, such as mitogenic responses (phytohemmagglutinin, concanavalin A, pokeweed mitogen), allogeneic mixed leukocyte reaction, antibody production and T cell colony formation in agar. In contrast, some BMMNC functions were elevated at low doses of IL-2 and IFN-gamma, and significant suppression of BMMNC was seen only with high doses of IL-2 (greater than or equal to 100 U/ml) and IFN-gamma (1,000 U/ml). IL-2 was by far more effective than IFN-gamma in both the amplification of NK activity and the suppression of most of the other functions. IL-2, but not IFN-gamma, was found to activate/induce suppressor cells and increased the proportion of Leu-2+ (CD8) cells in PBMNC; the suppressive effect was time- and dose-dependent. The IL-2-induced suppression could be diminished by inclusion of anti-IL-2 antibody during the pretreatment phase. Similar suppressive effects were noted in PBMNC from AIDS patients. These findings suggest that: (a) high-dose IL-2 may elicit immunosuppression which can be mediated by nondiscriminative highly cytotoxic cells (i.e. lymphokine-activated killer cells) and/or by noncytotoxic, nonspecific suppressor cells, and (b) that PBMNC respond differently to the lymphokines than do BMMNC.     

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.     

Lack of a central role for calcium in the induction and release of human interleukin-1

This study examines the potential role of calcium in the activation of human monocytes by bacterial lipopolysaccharide (LPS) to produce interleukin-1 (IL-1). Monocytes cannot be triggered to produce IL-1 through addition of calcium ionophores. Triggering of IL-1 production by LPS cannot be blocked by depletion of extracellular calcium, blockade of calcium channels, or addition of agents which antagonize the effects of intracellular calcium. Finally, the addition of LPS does not induce the mobilization of intracellular free calcium as measured by quin-2 fluorescence.     

Monocyte-mediated augmentation of human natural cell-mediated cytotoxicity

Normal human monocytes can significantly and rapidly augment natural cell-mediated cytotoxicity (NCMC) against K562 target cells. Approximately 50% augmentation was observed after direct mixture of monocytes with autologous null cells in the 4-hr chromium-release assay. This effect was dependent on the number of monocytes, and B cells and granulocytes were not effective. Coculture of null cells with monocytes and subsequent recovery of null cells for use as effector cells also produced significantly elevated cytolytic activity. This effect was dependent upon the number of monocytes, the length of time of coculture, and the cell donor. Augmentation of NK activity was rapid and observed after 0.5-12 hr of coculture, but suppression was observed after 36 hr; augmentation was observed with high monocyte:null cell (1:1, 1:2) ratios, and no effect was generally observed with lower ratios (1:8). At the single-cell level, the augmentation was associated with an increase in the proportion of target-binding cells which were lytically active. The augmentation of NK activity by monocytes required close cellular proximity, was mediated by a factor which was active or induced only in close proximity of the effector and producer cells, and/or was mediated by a soluble factor with a molecular weight greater than 50,000. This new demonstration that monocytes can augment as well as suppress NCMC may represent another avenue by which NK cell activity may be modulated in vivo.