Identification of a common signal associated with cellular proliferation stimulated by four haemopoietic growth factors in a highly enriched population of granulocyte/macrophage colony-forming cells.

We have prepared a population of bone marrow cells that is highly enriched in neutrophil/macrophage progenitor cells (GM-CFC). Four distinct haemopoietic growth factors can stimulate the formation of mature cells from this population, although the proportions of neutrophils and/or macrophages produced varied depending on the growth factor employed: interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulated the formation of colonies containing both neutrophils and macrophages; macrophage colony-stimulating factor (M-CSF) produced predominantly macrophage colonies; and granulocyte colony-stimulating factor (G-CSF) promoted neutrophil colony formation. Combinations of these four growth factors did not lead to any additive or synergistic effect on the number of colonies produced in clonal soft agar assays, indicating the presence of a common set of cells responsive to all four haemopoietic growth factors. These enriched progenitor cells therefore represent an ideal population to study myeloid growth-factor-stimulated survival, proliferation and development. Using this population we have examined the molecular signalling mechanisms associated with progenitor cell proliferation. We have shown that modulation of cyclic AMP levels has no apparent role in GM-CFC proliferation, whereas phorbol esters and/or Ca2+ ionophore can stimulate DNA synthesis, indicating a possible role for protein kinase C activation and increased cytosolic Ca2+ levels in the proliferation of these cells. The lack of ability of all four myeloid growth factors to mobilize intracellular Ca2+ infers that these effects are not achieved via inositol lipid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Keratinocyte-derived granulocyte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages

Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.     

Priming and triggering of tumoricidal and schistosomulicidal macrophages by two sequential lymphokine signals: interferon-gamma and macrophage cytotoxicity inducing factor 2

We describe a new lymphokine activity, macrophage cytotoxicity inducing factor 2 (MCIF2), in the T cell mitogen-induced supernatant of a murine T cell clone in long-term culture. MCIF2 has the following properties: it elutes from a Sephadex G-100 column in three m.w. forms (10, 34, and 100 KD); it is acid labile (pH 2 to 4) and heat sensitive (80 min at 56 degrees C); it is not constitutively secreted, coexists in the same supernatant with immune interferon (IFN-gamma), and synergizes with IFN-gamma for induction of tumoricidal and schistosomulicidal resident peritoneal mouse macrophages. We uncoupled this synergy and show that IFN-gamma serves as the first ("priming") and MCIF2 as the second ("triggering") signal for macrophage activation. Application of the lymphokines in the reverse order was ineffective. These data demonstrate a two-step mechanism of macrophage activation.     

Identification of a signal-transduction pathway shared by haematopoietic growth factors with diverse biological specificity.

The haematopoietic growth factors multi-colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2 specifically control the production and proliferation of distinct leucocyte series. Each growth factor acts on a unique surface receptor associated with an appropriate signal-transduction apparatus. In this report we identify a 68 kDa substrate which is phosphorylated after stimulation of different cell types with multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2. The 68 kDa substrate is also phosphorylated in each cell line stimulated with synthetic diacylglycerol, a direct activator of protein kinase C. Interestingly, granulocyte/macrophage colony-stimulating factor does not induce phosphorylation of the 68 kDa molecule. The 68 kDa molecule that is phosphorylated after stimulation with each ligand yielded similar peptide maps after chymotryptic digestion; furthermore, the substrate was always phosphorylated on threonine residues. Phosphorylation of the same residues in the 68 kDa substrate suggests that activation of protein kinase C is one common signal-transduction event associated with the action of multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2.     

Bactericidal effects of macrophages of mice treated with interferon]

The preparations of interferon or virus-inhibiting factor produced in L cell (L-IF) and mouse brain (MB-IF) enhanced the killing of Staphylococcus aureus (S.a.) by the mouse peritoneal macrophage. The L-IF, heat-inactivated at 80 degrees or 60 degrees for 30 min., and mock L-IF could not enhance the killing of S.a. The heterologous human and rabbit interferon preparations didn't enhance the bactericidal activity of macrophage. The L-IF didn't have any effect on the release of lysozyme from the macrophages.     

Identification and characterization of a B cell activation factor (BCAF) produced by a human T cell line

A human T cell line, Peer, that expresses the T cell helper phenotype produces discrete activation and growth factors for tonsillar B cells. The B cell activation factor produced by Peer is biochemically and physiologically distinct from other lymphokines known to enhance B cell proliferation, namely, interleukin 1, interleukin 2, interferon, and previously characterized B cell growth factors (BCGF). The BCGF produced by Peer is functionally similar to previously described BCGF but has a m.w. of approximately 30,000 daltons. The identification and characterization of a T cell-derived activation factor that can induce apparently resting (Go phase) B cells to enter S phase in the absence of an exogenous first signal has important implications in the additional dissection of the complex steps in the human B cell cycle.     

Macrophage colony-stimulating factor enhances monocyte and macrophage antibody-dependent cell-mediated cytotoxicity

In vitro culture of either human peripheral blood monocytes or murine peritoneal macrophages for 72 hr in the presence of macrophage colony-stimulating factor (M-CSF) dramatically increased their subsequent ability to mediate antibody-dependent cellular cytotoxicity (ADCC). The M-CSF-treated cells were more effective in ADCC at lower effector to target cell ratios and in the presence of lower concentrations of tumor-specific monoclonal antibody than the untreated control cells. Two other hematopoietic cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-3, reported to enhance other macrophage effector functions were ineffective in promoting the development of ADCC by cultured human monocytes. All three hematopoietic growth factors were capable of enhancing the ability of the cultured monocytes to secrete TNF alpha; however, TNF alpha is unlikely to be an important cytotoxic factor in ADCC because neutralizing antibodies against TNF alpha had no affect on ADCC in vitro. Further, much higher concentrations of M-CSF were required to augment monocyte TNF alpha release (20-100 ng/ml) than ADCC capacity (1-10 ng/ml). These results suggest that M-CSF administration might prove effective in increasing the tumoricidal activities of tumor-specific monoclonal antibodies by enhancing the capacity of monocytes and macrophages to mediate ADCC.     

Lipopolysaccharide-induced apoptosis of macrophages determines the up-regulation of concentrative nucleoside transporters Cnt1 and Cnt2 through tumor necrosis factor-alpha-dependent and -independent mechanisms

In murine bone marrow macrophages, lipopolysaccharide (LPS) induces apoptosis through the autocrine production of tumor necrosis factor-alpha (TNF-alpha), as demonstrated by the fact that macrophages from TNF-alpha receptor I knock-out mice did not undergo early apoptosis. In these conditions LPS up-regulated the two concentrative high affinity nucleoside transporters here shown to be expressed in murine bone marrow macrophages, concentrative nucleoside transporter (CNT) 1 and 2, in a rapid manner that is nevertheless consistent with the de novo synthesis of carrier proteins. This effect was not dependent on the presence of macrophage colony-stimulating factor, although LPS blocked the macrophage colony-stimulating factor-mediated up-regulation of the equilibrative nucleoside transport system es. TNF-alpha mimicked the regulatory response of nucleoside transporters triggered by LPS, but macrophages isolated from TNF-alpha receptor I knock-out mice similarly up-regulated nucleoside transport after LPS treatment. Although NO is produced by macrophages after LPS treatment, NO is not involved in these regulatory responses because LPS up-regulated CNT1 and CNT2 transport activity and expression in macrophages from inducible nitric oxide synthase and cationic amino acid transporter (CAT) 2 knock-out mice, both of which lack inducible nitric oxide synthesis. These data indicate that the early proapoptotic responses of macrophages, involving the up-regulation of CNT transporters, follow redundant regulatory pathways in which TNF-alpha-dependent- and -independent mechanisms are involved. These observations also support a role for CNT transporters in determining extracellular nucleoside availability and modulating macrophage apoptosis.     

Activation and proliferation signals in murine macrophages: stimulation of glucose uptake by hemopoietic growth factors and other agents

Purified colony-stimulating factor (CSF-1) (or macrophage colony stimulating factor [M-CSF]) stimulated the glucose uptake of murine bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM) as measured by 3H-2-deoxyglucose (2-DOG) uptake. Similar concentrations of CSF-1 stimulated the 2-DOG uptake and DNA synthesis in BMM. Other purified hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and interleukin-3 (IL-3) (or multi-CSF), and the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though differing in their mitogenic capabilities on BMM, were also stimulators of 2-DOG uptake in BMM and RPM. The nonmitogenic agents, lipopolysaccharide (LPS) and concanavalin A (Con A), were also active. The inhibition by cytochalasin B and by high concentrations of D-glucose suggest that the basal and stimulated 2-DOG uptake occurred via a carrier-facilitated D-glucose transport system. The responses of the two macrophage populations to the hemopoietic growth factors and to the other agents were quite similar, suggesting that events that are important for the induction of DNA synthesis are not tightly coupled to the earlier rise in glucose uptake. For the BMM, the ability of a particular agent to stimulate glucose uptake did not parallel its ability to promote cell survival. However, stimulation of glucose uptake could still be a necessary but insufficient early macrophage response for cell survival and subsequent DNA synthesis.     

Effect of cytokines on thymic hematopoietic precursors

Summary We have previously shown that the interaction of thymocytes with thymic accessory cells (macrophages and/or interdigitating cells) is one of the factors required for thymocyte activation. Precursors of both thymic accessory cell and thymocytes are included in the CD4^- CD8^- Mac-1^- Ia^- subpopulation, and their respective maturation and/or activation may be modulated by granulocyte-macrophage colony-stimulating factor, interleukin 1 and interleukin 2. When CD4^- CD8^- thymic cells are activated with granulocyte-macrophage colony-stimulating factor plus interleukin 2, both macrophages and interdigitating-like cells are present, as shown by electron microscopy. When activated with interleukin 1 plus interleukin 2, the interdigitating-like cells is the only accessory cell present. In both culture conditions, large clusters are formed between interdigitating cells and lymphoid cells. These results have led us to propose two-step signals for thymocyte proliferation: first, the maturation of macrophages under granulocyte-macrophage colony-stimulating factor control and the production of interleukin 1, and secondly, the maturation of interdigitating cells under interleukin 1 control, their clustering with thymocytes which are then activated.Abbreviations CFU-S colony-forming units in the spleen - CSF colony-stimulating factor - DC dendritic cells - DN double negative cells (CD4^- CD8^-) - EC epithelial cells - GM-CFC granulocyte/macrophage colony-forming cells - GM-CSF granulocytemacrophage CSF - IDC interdigitating cell - IL-1 interleukin 1 - IL-2 interleukin 2 - MØ macrophage - P-TR phagocytic cell of the thymic reticulum     

Normal mouse lung tissue produces a growth-inhibitory factor(s) preferential for mouse monocytic leukemia cells

Summary A growth-inhibitory (GI) factor, that specifically inhibits the growth of mouse monocytic leukemia cells, was found in conditioned medium of mouse lung tissue, but not in that of mouse brain, heart, liver, or kidney tissue. Conditioned medium of spleen or bone marrow cells had low GI activity. Pulmonary macrophages were as active as peritoneal and bone-marrow-derived macrophages in production of the GI activity. The GI factor inhibited the growth of murine monocytic leukemia cell lines Mm-A and J774.1, but scarcely inhibited the growth of other mouse cell lines, such as a myeloblastic leukemia cell line (M1), a Friend erythroleukemia cell line (745A) and a mammary carcinoma cell line (FM3A). It had no significant effect on the growth of human monocytic leukemia cell lines U937 and THP-1 or on the HL-60 promyelocytic leukemia cell line. These results suggest that the GI factor produced by mouse lung tissue preferentially inhibits the growth of mouse monocytic cells.The GI factor was found to be a proteinaceous substance with a molecular mass of 25 kDa. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 7.2–7.5. The GI activity was not significantly decreased by heat treatment at 56°C for 30 min or acid treatment (0.01 M HCl, 14 h), but the GI activity in glycosidase-treated conditioned medium of lung tissue was lost on heat treatment. The GI activity could not be neutralized with anti-(interferon + ) antibody. The activity was produced constitutively by lung tissues and its production was not stimulated appreciably by lipopolysaccharide, lectin, or poly(I)·poly(C). The GI factor appears to be a cytokine unrelated to known cytokines such as tumor necrosis factor, interleukin-1, transforming growth factor , and interferons. These results suggest that the GI factor may be involved in negative feedback regulation of macrophage production in steady-state conditions in the lungs.This work was supported in part by a grant for Cancer Research from the Ministry of Education, Science and Culture, and a grant from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control     

Properties of colony-stimulating factors produced by macrophage cell lines and hybrid cells

Colony-stimulating factors (CSFs) produced by two simian virus 40(SV40) transformed macrophage cell lines (BAM1 and BAM3), and three hybrids (HM3-11, HM3-12, and HM3-14) derived from fusion between BAM3 and a Chinese hamster cell line (hs222-16) were examined. HM3-11 and HM3-14 produce two molecular species of CSF, which are not found in the conditioned media from cultures of BAM1 and BAM3 or lipopolysaccharide (LPS), phorbolmyristate-acetate (PMA), and zymosan-stimulated BAM3. HM3-12, which is classified into another group in terms of CSF secretion, does not produce these two CSFs. On the basis of various criteria, one of these CSF species (peak 1-CSF) was characterized as a macrophage-colony-stimulating factor (M-CSF). The other CSF (peak 2-CSF) induced a group of bone marrow cells in granulocytes and macrophages as well as growth of a mast cell line, IC2. This CSF has an apparent molecular weight of 18,000, estimated by SDS-polyacrylamide gel electrophoresis. Unlike interleukin 3 (IL3) from WEHI-3 cells, the growth factor activity of peak 2-CSF binds to DEAE-Sephacel. Thus, peak 2-CSF is similar to a granulocyte-macrophage colony-stimulating factor (GM-CSF) rather than to IL3. The anti L cell CSF serum does not inhibit the CSF activity in Chinese hamster fibroblast conditioned medium, and the IC2 cells do not respond to Chinese hamster lung conditioned medium (CHLCM), suggesting that peak 1- and peak 2-CSF are of mouse origin.     

The differential time-course of extracellular-regulated kinase activity correlates with the macrophage response toward proliferation or activation

Bone marrow-derived macrophages proliferate in response to specific growth factors, including macrophage colony-stimulating factor (M-CSF). When stimulated with activating factors, such as lipopolysaccharide (LPS), macrophages stop proliferating and produce proinflammatory cytokines. Although triggering opposed responses, both M-CSF and LPS induce the activation of extracellular-regulated kinases (ERKs) 1 and 2. However, the time-course of ERK activation is different; maximal activation by M-CSF and LPS occurred after 5 and 15 min of stimulation, respectively. Granulocyte/macrophage colony-stimulating factor, interleukin 3, and TPA, all of which induced macrophage proliferation, also induced ERK activity, which was maximal at 5 min poststimulation. The use of PD98059, which specifically blocks ERK 1 and 2 activation, demonstrated that ERK activity was necessary for macrophage proliferation in response to these factors. The treatment with phosphatidylcholine-specific phospholipase C (PC-PLC) inhibited macrophage proliferation, induced the expression of cytokines, and triggered a pattern of ERK activation equivalent to that induced by LPS. Moreover, PD98059 inhibited the expression of cytokines induced by LPS or PC-PLC, thus suggesting that ERK activity is also required for macrophage activation by these two agents. Activation of the JNK pathway did not discriminate between proliferative and activating stimuli. In conclusion, our results allow to correlate the differences in the time-course of ERK activity with the macrophagic response toward proliferation or activation.     

Differentiation of Mouse Bone Marrow Precursor Cells into Neutrophil Granulocytes by an Activity Separation from WEHI-3 Cell-Conditioned Medium

Colonies comprised exclusively of neutrophil granulocytes have been obtained by growing mouse bone marrow cells in nutrient semisolid agar cultures. A stimulator of predominantly granulocyte colony formation was present in the breakthrough fraction of preparations of colony-stimulating activity separated on DEAE-Sephadex A. The source of colony-stimulating activity was concentrated conditioned medium of a murine myelomonocytic cell line (WEHI-3), which unfractionated stimulated the growth of colonies of granulocytes, macrophages, megakaryocytes, as well as mixed colony types. After stepwise column chromatography of the conditioned medium, the breakthrough fraction was shown to stimulate predominantly granulocyte colony formation, and the fraction eluted with 1 M NaCl was found to induce primarily macrophage colony growth. Colony morphology was independent of the concentration of eluate used. The morphology of colonies varied with increasing concentrations of the breakthrough fraction. At low concentrations, granulocyte colony formation was almost exclusively observed. With increasing concentrations of this fraction, an increasing proportion of the colonies were found to contain macrophages. The effect of concentration of this activity was in marked contrast to previous findings where the incidence of granulocyte colony formation was inversely related to the concentration of colony-stimulating activity. This differential responsiveness of cell to stimulus has previously been interpreted as low concentrations of a growth and differentiation factor being required for macrophage production and high concentrations of the same factor required for granulocyte formation. Separation of these activities by DEAE Sephadex chromatography, and alteration of the dose-response curve, such that granulocyte colony formation varies directly with the amount of stimulator, indicates that the differentiation of these two cell blood lineages may be controlled by separate entities.     

Production of B cell growth factor by normal human B cells

Although it has been demonstrated that malignant human B cell lines are capable of producing B cell growth factor (BCGF), production of BCGF by normal B cells has not been shown. In this study, we demonstrate BCGF production by normal B cells, achieved by using human peripheral blood B cells prepared by a positive selection technique and stimulated with Staphylococcus aureus Cowan I (SAC) for 12 hr. SAC was removed from the supernatants by anti-SAC-coupled Sepharose. Supernatants absorbed with this antibody were functionally free of SAC, as demonstrated by their inability to activate resting B cells. B cells stimulated with SAC for 12 hr produced BCGF activity that was generally unmeasurable in supernatants by 36 hr. Characterization of BCGF produced by SAC-stimulated B cells revealed a m.w. of 32,000 by high-performance liquid chromatography sieving and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; this BCGF was found to have an isoelectric point of 6.7. Furthermore, this BCGF lacked interleukin 1, interleukin 2, interferon, and B cell differentiation factor activity. This observation that BCGF can be produced by normal human B cells is significant because it demonstrates for the first time that normal B cells have the ability to provide their own growth factors or the growth factors for other B cells.     

Human macrophage-activating factors for cytotoxicity. I. Establishment of a human T-cell hybridoma that produces macrophage-activating factors for cytotoxicity

Human T cell hybridoma, H3-E9-6, that produces macrophage activating factors for cytotoxicity (MAF-C) was prepared by somatic fusion of phytohemagglutinin-activated peripheral blood lymphocytes with emetine/actinomycin D-treated cloned human acute lymphocytic leukemia cells (CEM 11). The activities of the following were assayed: (1) macrophage-activating factor for cytotoxicity of monocytes (MAF-C 1 day), (2) macrophage-activating factor for cytotoxicity of monocyte-derived macrophages (MAF-C 6 day), (3) macrophage-activating factor for cytotoxicity of murine macrophages (MAF-Cm), (4) macrophage-activating factor for glucose consumption (MAF-G), (5) macrophage-activating factor for O2- formation (MAF-O). The culture supernatant of H3-E9-6 showed MAF-C 1 day-MAF-C 6 day, MAF-Cm, and MAF-G activities. The MAF-Cm activity was considerably enhanced by the addition of murine recombinant interferon gamma (rIFN-gamma). The MAF-C 1 day activity in the H3-E9-6 sup was not decreased by heat treatment (56 C, 30 min), by pH 2 treatment or by the addition of monoclonal anti-human IFN-gamma antibody or polymyxin B. These data suggest that MAF-C in H3-E9-6 sup is distinct from human IFN-gamma or lipopolysaccharide (LPS).     

Macrophage-derived factors increase low density lipoprotein uptake and receptor number in cultured human liver cells.

Recent evidence suggests the possibility that macrophages can influence lipoprotein metabolism. Therefore we investigated the ability of cultured macrophages to alter low density lipoprotein (LDL) uptake in a human liver cell line (HepG2). Conditioned media from phlogogenic-induced mouse peritoneal macrophages or from a human macrophage cell line stimulated with endotoxin increased HepG2 LDL uptake by as much as 60-70%. The increase was due, in part, to a significant macrophage-induced 40% increase in the number of LDL receptors per cell. Although macrophage conditioned media inhibited HepG2 cholesterol synthesis, the LDL receptor up-regulation did not appear to be due to the effects on cholesterol synthesis. The LDL receptor stimulatory activity was sensitive to proteolysis and heat. Its molecular mass was approximately 20 kDa based on gel filtration. Several macrophage secretory proteins were tested in HepG2 cultures for LDL uptake stimulation. Of these, oncostatin M (approximately 18 kDa by gel filtration) gave the strongest response. The rank order for LDL uptake stimulation was oncostatin M much greater than interleukin 6 = interleukin 1 = transforming growth factor-beta 1. A neutralizing antibody directed against oncostatin M inhibited the ability of conditioned media to up-regulate LDL receptors by 85%. Thus, our results indicate that macrophages can secrete several proteins that up-regulate LDL receptors in HepG2 cells and that most of the up-regulatory activity in macrophage conditioned media appears to be due to oncostatin M.     

The Interaction of Acanthamoeba spp. with Activated Macrophages and with Macrophage Cell Lines

Acanthamoeba spp. are free-living amebae associated with amebic keratitis and chronic granulomatous amebic encephalitis. The present studies were undertaken to compare the pathogenicity of three species of Acanthamoeba in B₆C₃F₁ mice after intranasal challenge with Acanthamoeba-induced cytopathogenicity for different macrophage populations. The ability of murine macrophage cell lines and activated murine peritoneal macrophages to lyse Acanthamoeba has been assessed by coincubating macrophages with ³H-uridine labeled amebae. Conversely, destruction of macrophages by Acanthamoeba was determined by measuring the release of chro-mium-51 from radiolabeled macrophages. Acanthamoeba culbensoni , which is highly pathogenic for mice, destroys macrophage cultures in vitro. Activated primary peritoneal macrophages were more resistant to Acanthamoeba -mediated destruction than macrophage cell lines activated in vitro. Activated macrophages were capable of limited destruction of Acanthamoeba polyphaga and Acanthamoeba castellanii. Acanthamoeba -specific antibodies increased the amebicidal activity of activated macrophages. Macrophage-mediated destruction was by contact-dependent cytolysis and by ingestion of amebae. Conditioned medium obtained from macrophage cultures after treatment with lipopolysaccharide and interferon gamma was neither cytolytic nor cytostatic for Acanthamoeba spp. Purified recombinant cytokines including tumor necrosis factor α. interleukin 1α, and interleukin 1β, alone or in combination, were not cytolytic for Acanthamoeba trophozoites.     

Study of the biological activities of toxic shock syndrome toxin-1. I. Proliferative response and interleukin 2 production by T cells stimulated with the toxin

The mitogenic and interleukin 2 (IL 2) production-inducing effects of toxic shock syndrome toxin-1 (TSST-1) on murine lymphocytes were investigated. TSST-1, an exotoxin produced by Staphylococcus aureus recovered from patients with toxic shock syndrome (TSS), is thought to be a causative agent of the syndrome. TSST-1 was mitogenic for splenic T cells and peanut agglutinin (PNA)-negative thymocytes, but not for T cell-depleted spleen cells, PNA-positive thymocytes or IL 2-dependent CTLL 2-cells. A factor mitogenic for CTCC-2 cells with a molecular weight of 30-35 kdaltons was obtained by stimulating spleen cells with TSST-1 and it was absorbed by CTLL-2 cells, indicating that the factor is IL 2. For substantial amounts of IL 2 to be produced, 10 ng or more of TSST-1 per ml and 48 hr or more of incubation were required. Removal of T cells abrogated the IL 2 production by spleen cells. T cells obtained by the nylon wool column method alone produced IL 2 on TSST-1 stimulation in the presence of either macrophages or a macrophage lysate containing interleukin 1. However, T cells obtained by a combination of the nylon wool column method and anti-Ia antibody treatment produced IL 2 in the presence of macrophages but not of the macrophage lysate, indicating that IL 2 production by TSST-1-stimulated T cells is absolutely dependent on the presence of accessory cells.