Production of macrophage activating factor by human leukemic T cell lines

Human leukemic T cell lines were tested for their ability to produce a macrophage activating factor. When mouse peritoneal macrophages were cultured for 48 hr in the presence of culture supernatants from cell lines HPB-ALL, CCRF-CEM, or MOLT-4, glucose oxidation via the hexose monophosphate pathway was enhanced by five to seven fold. Culture supernatants from cell line HPB-MLT stimulated the oxidation to a lesser extent. However, cell line CCRF-HSB-2 was essentially inactive as a producer. The active supernatants also stimulated the release of hydrogen peroxide from macrophages, whereas the inactive one did not. Since treatment of the cell lines with 12-o-tetradecanoyl phorbol acetate or phytohemagglutinin had little effect on the production of the factor except HPB-ALL, the cell lines seemed to secrete the factor constitutively. The stimulatory effect was dose-dependent and evident at a concentration as low as a 1/80 dilution. The factor was resistant to heat treatment at 100 C for 20 min, nondialysable and sensitive to protease digestion. The activating factor could be partially purified by anion exchange and gel filtration chromatographies.     

Activation of human monocyte cytotoxicity by natural and recombinant immune interferon

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.     

Identification and characterization of a human T cell line-derived lymphokine with MAF-like activity distinct from interferon-gamma

Culture supernatants of several human T cell leukemia cell lines were screened for macrophage-activating activity (MAF) as defined by induction of tumoricidal activity against human melanoma cells in a 72-hr assay. Two cell lines, MT-2 and C10/MJ2, were found to produce high levels of MAF activity constitutively, but the MT-2 cell line, unlike C10/MJ2, produced little IFN-gamma. This observation was confirmed by Northern blot analysis performed with specific IFN-gamma cDNA probe. The MT-2 cell line thus provides a useful system to evaluate the existence of lymphokines with MAF activity that are distinct from IFN-gamma. The MAF activity produced by MT-2 cells was distinguished from IFN-gamma by the following criteria. MAF activity was not removed by immunoaffinity chromatography with the use of immobilized specific polyclonal antibodies to IFN-gamma and was not neutralized by a monoclonal antibody to IFN-gamma. Heat or acid treatments of IFN-gamma resulted in loss of its antiviral activity, but these treatments had no effect on MAF activity. MAF activity was not abolished by polymyxin B sulfate, suggesting that this activity is not mediated by or dependent on LPS. Initial characterization studies performed by using membrane filtration, gel filtration chromatography, and isoelectric focusing indicate that the non-IFN-gamma MAF activity produced by MT-2 cells has an apparent m.w. of 55,000 and an isoelectric point of 5.5. Collectively, these data suggest that the MT-2 human T cell line constitutively produces high levels of MAF and low levels of IFN-gamma and offers a useful source for the further purification of a unique human lymphokine with macrophage-activating activity that is distinct from IFN-gamma.     

An acidic lymphokine distinct from interferon-gamma inhibits the replication of herpes simplex virus in human pulmonary macrophages

Supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells were fractionated by gel filtration and isoelectric focusing. A fraction with an isoelectric point of 2.2-3.3 containing macrophage migration inhibition factor activity inhibited the replication of herpes simplex virus type 1 in human pulmonary macrophages and U937 cells. This fraction did not inhibit the replication of herpes simplex virus in human fibroblasts. Moreover, the ability of this lymphokine fraction to inhibit viral growth in macrophages was not neutralized by antibody against interferon-gamma. These findings identify a macrophage specific antiviral lymphokine which is distinct biochemically and immunologically from interferon-gamma.     

A novel human macrophage-activating factor: distinction from interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GMCSF)

Culture supernatant from a human T-cell leukemia virus type I (HTLV-1)-infected cell line, DGA-1, contained a novel macrophage-activating factor (MAF). This MAF was antigenically and functionally distinct from interferon-gamma (IFN-gamma) and from granulocyte-monocyte colony-stimulating factor (GMCSF). Potential contaminants such as bacterial lipopolysaccharide (LPS), Mycoplasma spp, and HTLV-1 were not responsible for this MAF activity. The DGA-1 MAF was secreted constitutively and the cell line grew well in the absence of growth factors such as interleukin-2, mitogen, or antigen. This cell line should provide a good source of this MAF for further purification and characterization.     

Macrophage-activating factor produced by a T cell hybridoma: physiochemical and biosynthetic resemblance to gamma-interferon

Biochemical and biosynthetic evidence has been obtained which indicates that the macrophage activating factor (MAF) produced by the murine T cell hybridoma clone 24/G1, which primes macrophages for nonspecific tumoricidal activity, is a form of gamma-interferon (IFN gamma). MAF and antiviral activities were generated by the clone in proportional amounts under a variety of culture conditions. Production of both activities showed identical dependence on cell density even when production precipitously ceased at cell concentrations greater than 1.2 X 10(6) cells/ml. MAF and antiviral activities displayed identical sensitivities to pH and temperature and were indistinguishable on the basis of binding to insolubilized polynucleotides. Dye-ligand chromatography of a stimulated hybridoma supernatant on a column of Matrex Gel Red A resulted in the 1500-fold purification and 100% recovery of the MAF activity. A qualitatively identical elution profile was also obtained for the antiviral activity; however, only 32% of the original activity was recovered. When subjected to gel filtration on a high performance liquid chromatography system (HPLC), the MAF and antiviral activities present in the Matrex Red pool displayed identical elution profiles and exhibited an apparent m.w. of 50,000. This technique resulted in another 10-fold purification and 50% recovery of the two activities. On HPLC chromatofocusing the MAF activity in the Matrex Red pool could be resolved into seven chromatographically distinct species yet could not be resolved from the antiviral activity on either a qualitative or quantitative basis. These results thus provide quantitative molecular evidence to support the concept that IFN gamma can act as a MAF.     

Characterization and purification of mouse macrophage procoagulant-inducing factor

Macrophage procoagulant-inducing factor (MPIF) is a product of mouse Lyt-1+2- cells that induces macrophage procoagulant activity (MPCA) on mouse peritoneal exudate cells or on the macrophage-like tumor cell line WEHI-265. Supernatants from Sepharose-bound concanavalin A-stimulated cells were fractionated by using DEAE-Sephacel, heparin-Sepharose, and isoelectric focusing. This procedure resolved three different MPIF: MPIF alpha (pI 8.5), MPIF beta (pI 8.8 to 9.2), and MPIF gamma (pI 5 to 5.5). MPIF alpha and beta were small molecules (approximately 14 kD and 20 to 25 kD) as determined by gel filtration on Sephadex G200 and Biogel P100. MPIF beta was sharply resolved as a peak eluting after lysozyme by gel filtration on HPLC columns I-150 and I-125, although SDS-PAGE of the HPLC-enriched material resolved two well-defined bands of 70 and 120 kD and some poorly defined material of 14 kD. Silver staining failed to detect components of MPIF alpha after SDS-PAGE. MPIF gamma activity was associated with material that separated over a broad range (20 to 60 kD and 60 to 200 kD), possibly due to aggregation with other components of the supernatants. Crude supernatants were stable to heating at 56 degrees C for 30 min and pH 2 treatment, although more highly enriched fractions were unstable to these treatments. Heating at 90 degrees C for 5 min totally destroyed MPIF activity. The properties of the two basic MPIF differ from other lymphokines known to affect macrophage function, e.g., colony-stimulating factor, migration-inhibition factor, interferon-gamma, and interleukin 1.     

Biochemical and functional characterisation of macrophage stimulating factors secreted by mitogen-induced goldfish kidney leucocytes

Mitogen-stimulated goldfish kidney leucocytes secrete a number of different macrophage activation factors (MAF) that induce profound physiological changes in macrophages. MAF produced by goldfish kidney leucocytes was characterised using fast performance liquid chromatography (FPLC) and bioassays that measured MAF-induced respiratory burst (RB) and nitric oxide (NO) responses of activated macrophages. Mitogen-induced fish kidney leucocyte supernatants were fractionated using gel permeation FPLC (GP-FPLC) and the ability of different fractions to induce NO or RB measured. A MAF of M(r) 50 kD, that induced a potent nitric oxide response in both a long-term goldfish macrophage cell line (GMCL) and in in vitro-derived fish kidney macrophages (IVDKM) was identified. The GP-FPLC partially purified 50 kD MAF activity occasionally induced significantly higher nitric oxide production than that of the crude MAF preparations. This increase in the NO-inducing activity was due to segregation of the 50 kD MAF from a novel macrophage deactivating molecule of M(r) 10-12 kD present in crude MAF preparations. This 10-12 kD molecule was shown to inhibit nitric oxide production in cytokine-activated goldfish macrophages. Mitogen-induced fish kidney leucocyte supernatants contained two distinct MAFs that induced the respiratory burst in GMCL and IVDKM: the 50 kD and 30 kD proteins. The partially purified 30 kD MAF primed goldfish macrophage for increased RB activity after only 6 h of treatment, and continued to augment the RB activity after 24 h of stimulation. In contrast, the GP-FPLC partially purified 50 kD molecule also primed the RB after only 6 h of stimulation, but subsequently deprimed the RB after 24 h of stimulation, an effect similar to that observed for crude MAF preparations. The 50 kD MAF activity was further purified using chromatofocusing FPLC (C-FPLC) using basic pH gradients and was shown to consist of two distinct NO-inducing molecules (> pI 9.3). Mitogen-stimulated fish kidney leucocytes secrete several factors that profoundly affect the anti-microbial responses of teleost macrophages and which undoubtedly are responsible for regulating teleost macrophage function in vivo.     

Suppressor cell induction factor: a new mediator released by stimulated human lymphocytes and distinct from previously described lymphokines

Suppressor cell induction factor (SIF) was produced by alloantigen-stimulated human peripheral blood lymphocytes, and it activated human T cells to become effective suppressors of the mixed lymphocyte reaction (MLR). The activity of SIF was resistant to 56 degrees C and to pH 2, and was precipitated by 50 to 80% saturated ammonium sulfate. SIF had a m.w. range, as determined by gel filtration, of 18,000 to 29,000; it did not bind to DEAE cellulose columns; and it was recovered in the pH range from 6.9 to 7.3 on isoelectric focusing. SIF was biochemically separable from IL 2, BF, IFN-gamma, and CSF. Furthermore, IL 2 activity was completely removed by absorption of MLC supernatants by murine cytotoxic T lymphocyte line (CTLL) cells, whereas SIF activity was unabsorbable, thus distinguishing SIF from IL 2. In addition, antiviral activity of MLC supernatants was completely abolished by anti-human IFN-gamma serum, whereas SIF activity was unaffected by this antiserum, thus distinguishing SIF from IFN-gamma. Since treatment of these supernatants with antiserum against human lymphoblastoid cell IFN(alpha/beta) had no effect on either antiviral or SIF activities in these supernatants, SIF was also distinguishable from IFN alpha/beta. These results indicate that SIF is a distinct new lymphokine with the ability to induce suppressor function in human T cells.     

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.     

A human macrophage hybridoma producing a cytotoxic factor distinct from TNF,LT, and IL-1

Summary A stable human macrophage hybridoma was established by somatic cell fusion between human peripheral blood monocyte-derived macrophages and an 8-azaguanine resistant clone of a human histiocytic lymphoma cell line U-937 (clone U-937-F9). The hybrid cell line (F9P) exhibited typical macrophage-like morphology and had 30 more chromosomes than U-937-F9 cells. Its macrophage characteristics were confirmed by the manifestation of intracellular nonspecific esterase, the detection of Mo-2 and LEU-M3 antigens on the cell surface, and the demonstration of phagocytic activity. Furthermore, when stimulated with lipopolysaccharide (LPS), this cell line could secrete a considerable amount of a cytotoxic factor (CTF). Distinct from the hybrid cell line, the parental U-937-F9 cells expressed neither Mo-2 nor LEU-M3 antigens on the cell surface, did not show phagocytic activity, and their culture supernatants did not show cytotoxic activity even after LPS stimulation. The activity of CTF in the culture supernatant of the LPS-stimulated hybrid cells could not be neutralized with anti-tumor necrosis factor, anti-interleukin-1, or anti-lymphotoxin antibodies. The CTF had a relative molecular mass of 45–60×10³ daltons as determined by gel filtration on a column of Superose 12, and an isoelectric point of 5.1. The cytotoxic activity was also induced when the hybrid cells were stimulated with the concentrated supernatants of a human T-cell hybridoma containing macrophage activating factor for cytotoxicity or with LP3 tumor cells which were used as target cells.     

Interferon-gamma is required in activation of macrophages for tumor cytotoxicity

The participation of interferon-gamma in activation of murine macrophages for tumor cell lysis was investigated. Biochemically macrophage activation factor and interferon-gamma have not been separated. Antiviral titers correlated closely with macrophage activation in antigen- or mitogen-induced spleen cell supernatants. A monoclonal rat antibody that neutralized virus-induced interferon was also found to neutralize interferon-gamma in such supernatants. These monoclonal antibodies were coupled to CH-Sepharose 4B and used for absorption of antiviral activity from mitogen-induced spleen cell supernatants. Absorption of the interferon was paralleled by the reduction of the macrophage-activating capacity of the supernatants. Data from control absorptions supported the specificity of the absorption effect. These results indicate that interferon-gamma is required for activation of macrophages for tumor cell lysis. These results can be interpreted in two ways: (a) the monoclonal antibodies cross-react with interferon-gamma and with a mediator that is required for activation of macrophages for tumor cell lysis or (b) interferon-gamma itself is an essential cofactor for macrophage activation.     

Functional analysis of cloned macrophage hybridomas. VII. Modulation of suppressor T cell-inducing activity

We have previously characterized a macrophage hybridoma clone, termed clone 59, which induced immunity but consistently failed to induce Ts responses. Macrophage 59 cells were cultured with supernatants from several activated T cell clones to determine if lymphokines could modulate the activity of this macrophage hybridoma to generate effector Ts. Culture supernatants from Th1 clones and from one atypical IL-4 and IFN-gamma-producing T cell clone successfully modulated clone 59 cells to induce effector Ts cells. In contrast, supernatants from activated Th2 cells failed to generate Ts-inducing activity in macrophage 59 cells. Culture with recombinant derived IFN-gamma was sufficient to cause modulation of Ts-inducing activity in macrophage 59 cells. The data imply that the differential functional activities ascribed to various macrophage hybridoma clones reflect macrophage heterogeneity instead of independent macrophage lineages. The suppression induced by clone 59 macrophages was genetically restricted to the putative I-J region. The ability of IFN-gamma containing supernatants to endow macrophage 59 with the capacity to induce effector suppressor cells was specifically abrogated by addition of an anti-IJk-idiotype antibody, which also reacts with IJ-interaction molecules, indicating that the mechanism of modulation most likely involves expression of IJ-interaction molecule determinants on antigen presenting cells.     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.     

Activation of rat and human alveolar macrophage intracellular microbicidal activity by a preformed LGL cytokine

An alveolar macrophage-activating factor was released from Percoll fractionated large granular lymphocytes (LGL) within minutes of contact with either the natural killer (NK)-sensitive K562 tumor or heat-killed Staphylococcus aureus. The factor enhanced the intracellular killing of S. aureus without altering the rate of phagocytosis. Factor release was blocked by treatment of LGL with monensin, a carboxylic ionophore that inhibits vesicular traffic, but was unaffected by actinomycin D and cycloheximide pretreatment, suggesting that the cytokine was performed. The cell producing the factor was found only in Percoll fractions containing high concentrations of lytic NK cells and LGL, and the phenotypes of the LGL were HNK-1+ and E rosette-. The macrophage activating factor was a small protein of 10,000 to 20,000 daltons, as determined by gel fractionation, and was sensitive to proteolytic enzymes and heat and pH labile. Active supernatants were devoid of antiviral (interferon; IFN) or interleukin 2 (IL 2) activity, and IFN-beta, IFN-gamma, IL 2, and interleukin 1 were unable to activate staphylococcidal activity, suggesting that the LGL macrophage activating factor was distinguishable from these cytokines.     

Alloreactive cloned T cell lines. VI. Multiple lymphokine activities secreted by helper and cytolytic cloned T lymphocytes

Culture supernatants generated by alloantigenic or lectin stimulation of a cloned helper T lymphocyte, designated L2, contain interleukin 2 (IL 2), granulocyte/macrophage colony-stimulating factor (CSF), B cell stimulating factor (BCSF), macrophage (Ia+)-recruiting factor (MIRF), (Ia+)-inducing activity, gamma-interferon, Fc receptor-enhancing activity, macrophage migration inhibitory factor (MIF), macrophage activation factor (MAF), interleukin 3 (IL 3), and a factor responsible for prolonging the synthesis and secretion of the fourth and second components of complement by guinea pig peritoneal macrophages. Erythropoietin was not detected. A spontaneously arising variant of L2, designated L2V, produces much lower quantities of macrophage-stimulating activities, IL 2, and interferon. However, when compared to L2, L2V produces much higher levels of BCSF, equivalent amounts of IL 3, and slightly smaller amounts of CSF. Unlike L2V, a cytolytic clone, designated L3, secretes lymphokines that primarily affect macrophage function. The time course of lymphokine production by L2 cells indicates that for the six lymphokine activities studied there are three different times at which maximal or near maximal levels are reached, as follows: 1) IL 2, 12 to 24 hr; 2) IL 3 and CSF, 24 to 48 hr; and 3) (Ia+)-inducing activity, MAF, and interferon, 48 hr or later. Only IL 2 activity disappears during the 8-day culture cycle. The time course data and the differential production of activities by the three types of lymphocyte clones suggest that at least four terminal effector lymphokine molecules account for the ten biologic activities tested.     

Purification and sequencing of glycosylation variants of BSF-1, as a MAF, from the EL-4 leukaemia cell line

Macrophage activation activity was characterized from a PMA-induced subclone of the murine EL-4 leukaemic cell line. The MAF was purified from the cell line culture supernatant by concentration, CM-Sepharose and lentil lectin Sepharose chromatography, AcA 54 gel filtration, Mono Q FPLC and reverse phase HPLC. Four protein bands of different abundance were observed on SDS-PAGE with molecular weights of 17,500 to 21,000 Da. Three of the four proteins were sequenced from the N-terminal and shared homology with the published sequence of BSF-1. Variation of the molecular weight due to glycosylation was demonstrated by N-glycanase treatment, all four proteins gave a band of 14,200 Da after deglycosylation. Both glycosylated and deglycosylated forms of BSF-1 were equally active in the MAF assay. A monoclonal antibody to BSF-1 neutralized 80% of the activity from crude culture supernatants in the MAF assay. These studies have indicated that BSF-1 is the major, if not the only, MAF activity from this particular subline of the murine EL-4 leukaemic cell line.     

Composition of splenic T lymphocytes in allophenic mice.

Guinea pig peritoneal macrophages were activated in vitro by culturing with MAF (macrophage activating factor)-containing fractions from stimulated lymphocytes. These macrophage preparations demonstrate a 60% increase in the production of prostaglandins of the E series (PGE) when compared with macrophages cultured with fractions from unstimulated lymphocytes. PGE accumulation in macrophage cultures is maximal after 24 hr with MAF; tumor cytotoxicity is also maximal at this time. The final PGE concentration in cultures of activated macrophages averaged 3 × 10^?8M.     

Lymphokine stimulation of human macrophage C2 production is partially due to interferon-gamma

Monocyte complement stimulator (MCS), a product of T lymphocytes, is defined by its ability to stimulate the synthesis and secretion of the second complement component (C2) by monocytes. Most macrophage-activating factor (MAF) activity present in lymphokine-rich culture supernatants has recently been found to be due to interferon-gamma (IFN-gamma). We therefore hypothesized that IFN-gamma may have MCS activity as well. We tested recombinant, E. coli-derived, human IFN-gamma (rIFN-gamma) for its effects on C2 production by adherent peripheral blood monocytes and U937 cells, a human monocytic cell line. Recombinant IFN-gamma in concentrations ranging from 0.1 to 300 U/ml (0.003 to 8.8 ng/ml) stimulates C2 production by both cell populations. Exposure of responding cells for at least 24 hr is required for maximal stimulation. To determine the contribution of IFN-gamma toward total MCS activity in crude lymphokine-rich supernatants, we employed a solid-phase immunoabsorption technique with the use of a monoclonal anti-IFN-gamma antibody. This technique removed all IFN-gamma detectable by a sensitive ELISA, but MCS activity was decreased by only 40 to 50%. Additionally, MCS activity of these supernatants did not correlate with IFN-gamma content as determined by ELISA. By using another method to eliminate IFN-gamma activity, acid dialysis destroyed all rIFN-gamma activity, as measured by stimulation of U937 C2 synthesis, but eliminated only 30 to 67% of MCS activity from crude lymphokine preparations. Thus IFN-gamma stimulates C2 production by monocytes and U937 cells and apparently accounts for some, but not all, MCS activity present in lymphokine-rich supernatants. Other lymphokines are present in such supernatants that also possess this activity.     

Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.