The mechanism of cell surface changes of guinea pig macrophages activated with purified migration inhibitory factor/macrophage activation factor

Guinea pig MIF (MIF/MAF), which was purified by immunoadsorbent column chromatography using an antibody against MIF/MAF, was observed to induce characteristic cell surface changes in macrophages under scanning electron microscopy (SEM). MIF/MAF induced enlarged petal-like ruffles in both rounded and spreading macrophages. The changes were observed as early as 2 hr after stimulation with MIF/MAF and continued for 24 hr. These morphological changes appeared to be a good indicator of macrophage activation and migration inhibition in the early phase. The mechanism of the characteristic ruffle formation was studied using metabolic inhibitors and reagents known to affect microfilaments and microtubules. When macrophages were treated with MIF/MAF in the presence of mitomycin C, actinomycin D, or puromycin, formation of the petal-like ruffles was not affected. However, vinblastine and cytochalasin B inhibited the induction of these ruffles. These results indicate that microtubule and microfilament assembly, but not synthesis of DNA, RNA, and protein, are required for the formation of the petal-like ruffles. In addition, treatment with a Ca²⁺ ionophore induced the same petal-like ruffles in macrophages, while treatment with dibutyryl-cyclic AMP or-cyclic GMP did not. These findings suggest that Ca²⁺ plays an important role in macrophage activation by MIF/MAF, especially in the early phase.     

Partial purification of guinea pig MIF by affinity column chromatography using macrophages.

First we have confirmed the previous observation that the macrophage migration inhibitory factor (MIF) was adsorbed on normal peritoneal macrophages when they were incubated at 4 C for 60 min. It was found that macrophages fixed with 2% glutaraldehyde gave more reproducible results than viable cells in terms of "adsorption" of guinea pig MIF. The adsorption was achieved more completely at 37 C than at 4 C, indicating that this reaction is a temperature-dependent phenomenon. Using these glutaraldehyde-fixed macrophages, a kind of cell-affinity column was successfully developed. The guinea pig MIF preparation lost its activity when it was passed through this affinity column, and MIF adsorbed on the column was recovered by elution with 0.1 M (L)-fucose of 0.1 M (D)-glucose. Such MIF active eluate was found to be at least 30--40 fold more pure than the original MIF preparation which had been previously fractionated according to its molecular weight. Therefore, this type of macrophage-affinity column may be useful for the purification of MIF.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity: I. Suppression of cutaneous delayed-type hypersensitivity by migration inhibitory factor

The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent column by using anti-guinea pig MIF antiserum. Suppression of cutaneous delayedtype hypersensitivity was achieved by intraperitoneal injection of the MIF fraction into the animals bearing macrophage-rich peritoneal exudates. Skin reactions induced by phytohemagglutinin (PHA) were also suppressed in these animals. Reactivity to skin reactive factor (SRF) was suppressed in these animals as well. The sera obtained from these animals exhibited the inhibitory activity against production of lymphokines from sensitized lymphocytes.     

Cell surface receptors for lymphokines. I. The possible role of glycolipids as receptors for macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF).

Incubation of culture supernatants from concanavalin A-stimulated guinea pig and rat lymphocytes with protein-free preparations of bovine brain gangliosides abolished their macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF) activity. The identity of the MIF/MAF-binding component(s) present in these glycolipid mixtures has yet to be established, but adsorption experiments using purified preparations of mono- (GM₁, GM₂, and GM₃), di- (GD_1a), and trisialogangliosides (GT₁) were negative. Since these gangliosides account for over 90% of the glycolipid content in brain ganglioside mixtures it appears that the MIF-binding component(s) is present only in very small amounts. Treatment of guinea pig peritoneal macrophages with liposomes containing similar brain gangliosides or water-soluble glycolipids extracted from guinea pig macrophages enhanced their responsiveness to MIF. The enhanced response to MIF of liposome-treated macrophages was abolished by incubation of the treated macrophages with fucose-binding lectins (Lotus agglutinin and Ulex europaeus agglutinin I) before exposure to MIF, suggesting that the MIF-binding component donated by the liposomes may be a fucose-containing glycolipid. The possible role of glycolipids as surface receptors for MIF and MAF is discussed.     

Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells

Experiments were conducted to purify the differentiation-inducing factor (D-factor), which induces differentiation of mouse myeloid leukemic cell line, Ml, into macrophage-like cells, in a conditioned medium of guinea pig peritoneal macrophages stimulated with lipopolysaccharide. On gel filtration under high performance liquid column chromatography (HPLC), D-factor eluted at the position of 45-15 KD. By the subsequent separation on DEAE HPLC the D-factor activity disappeared. However, in the presence of recombinant human IL 1 alpha the D-factor activity appeared at a position where tumor necrosis factor (TNF) eluted. Even after fractionation on hydroxyapatite HPLC the IL 1-dependent D-factor was co-chromatographed with TNF. Recombinant human TNF as well as the partially purified guinea pig TNF induced differentiation of Ml cells in conjunction with either the partially purified guinea pig IL 1 or recombinant human IL 1 alpha, although these factors by themselves did not induce differentiation. These findings suggest that a part of D-factor activity in the conditioned medium resulted from the cooperative effects between TNF and IL 1.     

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.     

d-Mannose as a component of the macrophage surface receptor for macrophage-activating factor (MAF) in mice

Mouse peritoneal exudate macrophages were rendered cytostatic and cytolytic for various mouse tumor cells in vitro by exposure to partially purified lymphokines containing macrophage-activating factor (MAF) at 37 °C for 2 hr. The macrophage activation disappeared completely when either 0.1 Md-mannose or 0.1 M methyl-d-mannoside was present with MAF. On the other hand, neither d-galactose nor d-glucose inhibited the activation, and l-fucose, l-rhamnose, and N-acetyl-d-glucosamine inhibited it only partially. Incubation of either macrophages or MAF with 0.1 Md-mannose for 2 hr had no effect on activation of the macrophages by the MAF. Treatment of the macrophages by α-d-mannosidase rendered them no longer responsive to MAF. Macrophages treated by either neuraminidase or proteolytic enzymes, but not with β-d-galactosidase lost their ability to respond to MAF. Treatment of MAF with α-d-mannosidase did not affect MAF activity. In addition, MAF activity was not reduced by passage through a column of immobilized concanavalin A. In an absorption experiment, the presence of d-mannose was shown to prevent the adsorption of MAF to macrophages, while d-galactose did not. Treatment of macrophages with plant lectins having affinity for d-mannose, sialic acid or l-fucose prevented the adsorption of MAF, but the other lectins did not. Mouse MAF failed to adsorb to guinea pig peritoneal exudate macrophage, which were suggested as having a fucose-containing glycolipid as a lymphokine receptor. Taken together, these results strongly suggest that the receptor for MAF on mouse macrophages may be a glycoprotein containing d-mannose and sialic acid as essential components.     

Characterization of a macrophage chemotactic lymphokine produced by purified protein derivative stimulation in vitro and in vivo

Using an immunoadsorbent column conjugated with anti-macrophage chemotactic factor-c (anti-MCF-c), MCF-c which has been separated and highly purified from a delayed-type hypersensitivity reaction (DHR) site, shares common antigenicity with the major macrophage chemotactic lymphokine released from purified protein derivative (PPD)-stimulated lymphocytes and also macrophage chemotactic lymphokine from phytohemagglutinin (PHA)-stimulated lymphocytes. Using a combination of the immunoadsorbent column and Sephadexgel chromatography these two lymphokines are purified to homogeneity from PPD- or PHA-stimulated guinea pig lymphocyte culture supernatants. These observations, taken in conjunction with the similarity in biological activities, physicochemical properties, and antigenicities, suggest that these two mediators are very similar, or possibly identical. MCF-c with chemotactic activity for macrophages seemed to exist as complexes with serum protein at the skin site of PPD-induced DHR in guinea pigs. The active substance, separated from the complexes under acid conditions, is indistinguishable from the major macrophage chemotactic lymphokine released by PPD stimulation with respect to antigenicity, heat stability, and non-diffusibility. They both have a molecular weight of about 12, 500. The chemotactic lymphokine formed comparable complexes with serum protein under neutral conditions; however, this complex dissociated in acid without loss of activity.     

Chemical modification of macrophages enhances their response to human macrophage activation factor

Human monocyte-derived macrophages pretreated with the esterase inhibitors, antithrombin III and soybean trypsin inhibitor (STI) showed increased responsiveness to limiting concentrations of macrophage activation factor (MAF) as demonstrated by their enhanced cytotoxicity for tumor cells. Other proteins that are not esterase inhibitors did not enhance the effect of MAF on the macrophages. Enhancement of MAF activity was also obtained when macrophages were preincubated with the cell surface reactant diazotized sulfanilic acid (DSA). When MAF was preincubated with untreated, DSA-treated or STI-treated macrophages prior to testing on fresh macrophages, MAF preincubated with the untreated macrophages lost activity whereas the cytotoxicity induced by MAF preincubated with DSA-treated or STI-treated macrophages showed no decrease. These findings suggest that the enhanced responsiveness to MAF is the result of decreased destruction of MAF by macrophage-associated proteinases. Thus, the response of guinea pig and human macrophages to MAF appears to be regulated by similar mechanisms.     

Mastocytoma cell migration in vitro: inhibition by MIF-containing supernatants.

Supernatants with macrophage migration inhibition factor (MIF) activity were obtained from cultures of antigen-stimulated guinea pig and human lymphocytes, and from SV40-infected monkey kidney cells. The monkey and human but not guinea pig preparations were effective in inhibiting migration of mastocytoma cells as well as macrophages. This inhibition of migration was not associated with cytotoxicity and was reversible.     

Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines

Reversible inhibitors of protein synthesis, cycloheximide and puromycin, and an irreversible inhibitor of RNA synthesis, actinomycin D, were employed to study the kinetics and types of macromolecular synthetic events required for the production of migration inhibitory factor (MIF) and macrophage activating factor (MAF) by Con A-stimulated lymphocytes. Reversible inhibition of protein synthesis during the first 2 hr of stimulation completely inhibited MIF and MAF production. The same treatment, performed 4 hr after the beginning of the stimulation, had no effect. When the inhibitors of protein synthesis were left in the cultures, a block of lymphokine production was observed when the drugs were added at 6 hr as well as at time 0. In contrast, irreversible inhibition of RNA synthesis at 6 hr was ineffective and only treatment at the beginning of culture blocked lymphokine production. These data suggest that a critical protein is synthesized during the first few hours of stimulation, which is required for subsequent production of lymphokines. After this special early requirement, however, continued protein synthesis is needed for lymphokine production. In contrast, the RNA required for MIF and MAF production seemed to be completely synthesized within 4 to 6 hr of stimulation. The possibility that suppressor macrophages inhibit lymphokine production via modulation of macromolecular synthesis is discussed.     

Possible role of macrophage glycolipids as receptors for migration inhibitory factor (MIF).

Guinea pig peritoneal exudate cells incubated with water soluble glycolipids obtained from macrophages show an enhanced response to migration inhibitory factor. Incorporation of these glycolipids into liposomes greatly facilitates their interaction with indicator cells. Enhancement of peritoneal exudate cell responsiveness to migration inhibitory factor was specific for glycolipids from guinea pig macrophages. Glycolipids extracted from guinea pig brain and polymorphonuclear leukocytes as well as several bovine and porcine glycolipids had no effect. Specificity of enhancement was not due merely to a preferential association of macrophage glycolipids with indicator cells. The possible role of macrophage glycolipids as receptors for MIF is discussed.     

Release of macrophage migration inhibitory factor(s) from lymphocytes stimulated by streptococcal preparations

Lymphocytes from apparently healthy subjects, incubated for 5 hours with cellular components or extracellular products of group A streptococci and then washed and reincubated, were found to release factor(s) capable of inhibiting guinea pig lung macrophage migration (“indirect method”). Inhibitition of macrophage migration was also obtained when the same preparations were tested directly on guinea pig lung cells, a macrophage-lymphocyte population (“direct method”). The guinea pigs had not been experimentally sensitized. The inhibition of migration appeared to depend on the presence of lymphocytes among the macrophages, since macrophages purified by repeatedly discarding nonadherent cells proved resistant to the migration inhibiting activity of the most active Streptococcal preparation, a 20 × concentrated filtrate. Reconstitution of the original lymphocyte-macrophage mixture reestablished the reactivity. The macrophage migration inhibition did not correlate with the age of the guinea pigs. It could not be obtained with preparations of group D streptococci or of Salmonella paratyphi. Group C streptococci did not inhibit the macrophage migration with the indirect method, but it did with the direct one.The factor(s) released into the medium on stimulation of apparently normal lymphocytes by Streptococcal preparations was relatively heat resistant, nondialyzable, and DNase and RNase resistant; its release was inhibited by puromycin. Pretreatment of the cells with trypsin prevented the absorption of the factor(s) and left migration unaffected. These characteristics are similar to those previously described for the migration inhibitory factor (MIF) produced by the interaction of sensitized lymphocytes and specific antigens. Whether or not these similarities indicate an identity remains to be determined.     

Glycolipid-dependent interaction between human migration-inhibitory factor and mononuclear phagocytes

It has been previously established in the guinea pig that the response of peritoneal macrophages to migration inhibitory factor (MIF) is enhanced by a macrophage glycolipid and that gangliosides reversibly bind MIF. This suggests that glycolipids function as cell surface receptors for MIF. In this report, it is demonstrated that the response of human peripheral blood monocytes to human MIF is augmented by preincubation of these cells with glycolipidenriched material extracted from the human macrophage-like cell line U937 or human peripheral blood monocytes and with a purified glycolipid from guinea pig peritoneal macrophages. In addition, a mixed ganglioside preparation from bovine brain shows the same effect. In contrast, the pure gangliosides, G_M1 and G_D1a, and glycolipids from the HL-60 cell line, which is a MIF-unresponsive cell line, were not able to enhance the response to human MIF. The specificity of enhancement by particular glycolipids could not be attributed to an increased uptake of only enhancing glycolipids since there was no significant difference between the association of monocytes with radioactive liposomes containing biologically active or inactive glycolipids. Pronase treatment did not affect the enhancing activity of the U937 glycolipidenriched material. Incubation of cells with glycolipids results in enhancement only if done at 37 °C and not at 4 °C. Therefore, the association of lipid with the monocyte surface appears to be dependent on temperature.Further evidence for the receptor nature of these enhancing glycolipids is provided by experiments involving affinity purification experiments. Coupling of bovine brain mixed gangliosides to agarose resulted in a matrix capable of reversibly binding MIF. G_D1a-agarose was inactive in this respect.     

Purification of guinea pig tumor necrosis factor (TNF): comparison of its antiproliferative and differentiative activities for myeloid leukemic cell lines with those of recombinant human TNF

Recently, we suggested that the effect of differentiation inducing factor (D-factor) which is found in the supernatant of macrophages, and induced the differentiation of a mouse myeloid leukemic cell line, M1, into macrophage-like cells, may be a result of the cooperative effects of tumor necrosis factor (TNF) and interleukin 1 (IL-1). In this study, we purified guinea pig (G.P.) TNF secreted from peritoneal macrophages and compared the antiproliferative and differentiative effects of the G.P. TNF with those of recombinant human TNF (rHuTNF). The purification scheme consisted of ultrafiltration, gel filtration-high performance liquid chromatography (HPLC), DEAE-HPLC, and reverse-phase HPLC. The cytotoxic activity of the purified substance was approximately 1.5 x 10(8) U/mg. The isoelectric point was 5.2. The molecular weight was 40 to 45 kDa as estimated by gel filtration and 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence was determined to be Ser-Ala-Ser-Gln-Asn-Asp . . . . Approximately 76 or 71% homology between G.P. TNF and mouse or human TNF exists in the NH2-terminal 21 residues. The purified G.P. TNF and rHuTNF demonstrated D-factor activity only in the presence of recombinant human IL-1 alpha in M1 cells. We also determined the effect of TNF on two human myeloid leukemic cell lines (THP-1 and U937). The purified G.P. TNF and rHuTNF inhibited the growth of U937 cells, but did not induce their differentiation. In THP-1 cells, TNF slightly inhibited the growth and induced differentiation. In mouse cell lines G.P. TNF was more effective than rHuTNF for differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biochemistry and in vitro activity of soluble factors of activated lymphocytes

Summary Activated lymphocytes release numerous products which are either synthesized de novo or in increased amounts; some of these products play a role in the regulation of the immune response and are designated as mediators of cellular immune reactions or lymphokines. The first lymphokine described was the macrophage migration inhibitory factor (MIF) which has been studied most extensively with regard to its chemical and biological properties. Using sensitive radiolabelling techniques and an antiserum against highly purified fractions of MIF we were able to identify several products of activated guinea pig lymphocytes with different molecular weights of 15.000, 30.000, 45.000, 60.000 which all had an isoelectric point of 5.2 and were all inhibitory to macrophage migration. It is suggested, that these molecules are oligomers of a common subunit of molecular weight 15.000. It was further shown, that molecules of the same physical-chemical and serological characteristics are produced by activated B-cells, L₂C leukemia cells and growing fibroblasts, thus further substantiating earlier reports on the production of MIF by lymphoid and non-lymphoid cells. The described molecules were also shown not to contain determinants of the major histocompatibility complex and to be distinct from lymphotoxin, another lymphocyte activation product. It is concluded, that MIF is not sa single molecule but rather a system of structurally related molecules. Thier interaction with macrophages and possible relationship to macrophage activating factor is discussed.     

Inhibition of macrophage migration by virus-induced interferon preparations.

It was found that a preparation of mouse L cell interferon induced by Newcastle disease virus (NDV) possessed not only interferon activity but also inhibitory activity upon migration of guinea pig peritoneal macrophages (MIF activity). These activities were also observed in a preparation of human leukocyte interferon induced by NDV. The interferon and MIF activities shared common characteristics in the dose response, time course of in vitro production, thermal stability, sensitivity to trypsin and periodate, and elution pattern in CM-Sephadex column chromatography. However, gel filtration pattern with Sephadex G-100 showed two separate peaks. Fractions collected from the first peak, corresponding to a molecular weight of about 45 000, had only the MIF activity, while those collected from the second peak, corresponding to a molecular weight of about 30 000, had both the interferon and MIF activities. A preparation of mouse brain interferon induced by Japanese encephalitis virus had a much weaker MIF activity than the L cell interferon, although these preparations were equal in interferon activity (5000 units/ml).     

Effects of L2C leukemia on macrophage-mediated responses

Summary Preliminary experiments have suggested that guinea pig L₂C B-cell leukemia cells were able to evade macrophage-mediated lysis. To determine whether the L₂C cells were resistant to macrophage cytotoxic activity or whether factors associated with the L₂C leukemia contributed to a generalized inhibition of macrophage cytotoxic activity, pulmonary macrophages from strain 2 guinea pigs with L₂C leukemia were tested for their ability to lyse the susceptible K562 cell line after activation by lipopolysaccharide (LPS) or lymphokines. In addition, the potential presence of soluble inhibitors of macrophage tumoricidal activity in serum-free culture supernatants and in serum from strain 2 guinea pigs terminally ill with the leukemia was tested by determining the effects of leukemic guinea pig serum (LGPS) or L₂C-conditioned medium (CM) on the tumoricidal activity of normal pulmonary macrophages. Macrophages from guinea pigs terminally ill with L₂C leukemia were demonstrated to be depressed in their cytotoxic activity against the K562 cell after stimulation by either LPS or lymphokines when compared to normal macrophages. The lymphokine-stimulated cytotoxic activity of normal macrophages was inhibited in the presence of LGPS or CM. Oxidative burst activity of normal macrophages, as measured by zymosan-stimulated production of superoxide and hydrogen peroxide, was also inhibited under these conditions. The data presented here suggests that soluble factors associated with L₂C leukemia cells can suppress oxidative burst activity of macrophages in vitro and that this effect may contribute to the ability of the leukemia cells to evade macrophage-mediated cytotoxicity.     

Increased responsiveness to macrophage-activating factor (MAF) after alteration of macrophage membranes.

Guinea pig macrophages pretreated with the esterase inhibitor, antithrombin III (AT III) show increased responsiveness to macrophage-activating factor (MAF) as demonstrated by their enhanced cytotoxicity for tumor cells. Other proteins that are not esterase inhibitors did not enhance the effect of MAF on the macrophage. Enhancement of MAF activity was also obtained when macrophages were preincubated with the cell surface reactant, diazotized sulfanilic acid (DSA). These studies indicate that the effect of MAF can be enhanced by chemical modifications of the macrophage membrane. They also provide further evidence to support the hypothesis that an esterase on the macrophage membrane modulates this cell's responsiveness to lymphocyte mediators.     

Functional activation of immune lymphocytes by antigenic stimulation in cell mediated immunity: I. Requirement for macrophages in antigen-induced MIF production by guinea pig immune lymphocytes in vitro

The role of macrophages in the process of antigen-induced production of mediators in cellular immune response was studied, using the antigen-induced production of migration inhibitory factor (MIF) as a measure of the activation of immune lymphocytes.The production of MIF by guinea pig immune lymph node cells in response to the stimulation with PPD was abolished when the lymph node cells were depleted of adherent cell population by passing the cells through a Tetron fiber column and incubating the effluent cells in plastic dishes. These purified immune lymphocytes did not respond to particle-bound PPD, either. However, the response was obviously restored by the addition of a small number of the purified peritoneal adherent cells (macrophages) which had been pulse-treated with PPD. The PPD-pulsed macrophages produced no MIF by themselves. Thus, the results clearly indicated the requirement for macrophages in the process of antigen-induced MIF production by immune lymphocytes. Destruction of PPD-pulsed macrophages by freezing and thawing or by homogenization abrogated their ability to stimulate immune lymphocytes. Attempts to restore the response of the purified immune lymphocytes to PPD by adding 2-mercaptoethanol or the culture supernatant of macrophages to the medium have so far been unsuccessful.