Role of lymphokines in regulation of macrophage differentiation

The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages.     

Chemical treatment of macrophages increases their responsiveness to migration inhibitory factor (MIF).

The response of guinea pig macrophages to migration inhibitory factor (MIF) is altered by several chemical treatments. Treatment of macrophages with the diazonium salt of sulfanilic acid (5 x 10(-6) to 4 x 10(-4) M) significantly increases the response of these cells to MIF. Treatment with acetic anhydride also augments the response of these cells to MIF. The latter finding suggests that alteration of amino, hydroxyl, or sulfhydryl groups is involved in this phenomenon. Treatment of macrophages with sodium periodate (2 x 10(-5) to 10(-3) M) which is known to oxidize cis-glycols and with hydroxylamine (2 x 10(-5) to 2 x 10(-3) M), which reacts with carbonyl groups also increases response to MIF. The following experiments suggest that the significant alteration occurs at the level of the cell surface. Incubation of macrophages with the diazonium salt of sulfanilic acid at 4 degrees C, at which temperature pinocytosis is largely inhibited, is sufficient to increase the MIF response. The activity of the cytoplasmic enzyme aspartate aminotransferase, which in homogenates is susceptible to inactivation by low concentrations of the diazonium salt of sulfanilic acid, is not decreased when intact macrophages are incubated with high concentrations of the diazonium salt of sulfanilic acid. Cumulatively, these findings suggest that modification of different functional groups on the macrophage surface causes the same physiologic effect.     

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.     

Inhibition of Macrophage Migration by Virus-Induced Interferon Preparations1

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).     

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).     

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.     

Structural studies of migration inhibitory activity induced during murine tumor allograft rejection.

Lymphocytes from mice rejecting a tumor allograft produce a soluble activity that inhibits the migration of murine macrophages. The present studies show that this activity is stable with 56 °C heating for 30 min, is inactivated by trypsin, has a molecular size of about 45,000, and has an isoelectric point of 5.0–5.5. In addition, it does not inhibit the migration of guinea pig macrophages. These results indicate that this lymphokine is similar in its physicochemical properties to murine, guinea pig and human migration inhibitory factors (MIF) studied previously, but they also suggest that there may be structural differences in the functionally active portions of MIF from different species.     

Macrophage glycolipid receptors for human migration inhibitory factor (MIF): differentiated HL-60 cells exhibit MIF responsiveness and express surface glycolipids which both bind MIF and convert nonresponsive cells to responsiveness

The human promyelocytic leukemia line HL-60 when treated with a phorbol diester (TPA) differentiates into cells (HL60-TPA) that respond to human migration inhibitory factor (MIF). Unresponsive HL-60 cells became responsive to MIF when preincubated with a glycolipid-enriched preparation extracted from HL60-TPA cells, human monocytes, human macrophage-like (U937) cell line, or with the purified glycolipid receptor for MIF from guinea pig peritoneal macrophages. Human blood monocytes exhibited an increased response to MIF when preincubated with glycolipids from HL60-TPA and U937 cells but not from HL-60 cells. Finally, glycolipids from HL60-TPA cells but not from HL-60 cells were able to reversibly bind MIF when covalently coupled to agarose. These studies suggest that TPA induces the differentiation of HL-60 cells into MIF-responsive cells through the expression of a glycolipid receptor for MIF.     

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.     

Partial characterization of murine migration inhibitory factor (MIF).

These studies describe the production of murine migration inhibitory factor (MIF)3 in sufficient quantities to allow its partial characterization by physiochemical and enzymatic methods. MIF was obtained from murine spleen cell cultures (C57BL/6 strain) stimulated with concanavalin A (Con A). Characterization of murine MIF was performed using Sephadex G-100 gel chromatography, isopycnic centrifugation in a CsCl density gradient, polyacrylamide disc electrophoresis, heat stability, and enzymatic treatment. MIF-containing and control fractions were assayed on normal C57BL/6 peritoneal exudate cells by using a microcapillary tube assay. Peak MIF activity was found in a Sephadex G-100 fraction containing molecules the size of albumin and slightly smaller, molecular weight 67,000 to 48,000. Murine MIF was stable to heating at 56 degrees C for 30 min but lost its activity at 80 degrees C for 30 min. Incubation of G-100 fractions containing MIF with water insoluble chymotrypsin destroyed the activity of MIF, indicating its protein nature. CsCl density gradient centrifugation revealed that murine MIF had a buoyand density greater than protein, consistent with its being a glycoprotein. Further, when subjected to disc electrophoresis on polyacylamide gels, murine MIF migrated in a region cathodal to albumin. Thus, mitogen stimulation of murine spleen cells produced MIF in quantities which allowed its partial characterization and purification, and its comparison with human and guinea pig MIF; this makes it feasible to analyze the role of murine MIF in cellular immunity and in its relationship to lymphocyte mediators which regulate humoral immune responses.     

Transfer of phosphatidylcholine between different membranes in Tetrahymena as studied by spin labeling.

A permeability factor was extracted in a latent form from guinea pig skin and separated by ammonium sulfate fractionation into the pseudoglobulin fraction (30--50% saturation). The activation of the latent form of the permeability factor seemed to be caused in the desalting step by gel filtration with Sephadex G-50. The factor was partially purified by streptomycin treatment and column chromatography using hydroxyapatite, diethylaminoethyl cellulose and Sephadex G-75, in this order. Gel filtration showed that its molecular weight was approx. 35000. Its permeability activity was heat stable at 61 degrees C for 60 min at neutral pH, resistant at pH 5--10 and at ionic strengths from deionized water to 1 M NaCl at 4 degrees C. Its activity was transient and suppressed by guinea pig serum, but insensitive to an anti-histamic agent (triprolidine). Furthermore, its permeability activity was inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor and leupeptin, and completely adsorbed by soybean trypsin inhibitor affinity column. These findings suggested that the permeability factor was a serine-type protease.     

Fractionation of murine macrophage inhibitory factor into two distinct species

Murine migration inhibitory factor (MIF) produced by concanavalin A-stimulated lymph node cells from C57BL/6 mice was fractionated by Sephadex G-100 gel filtration, density gradient electrophoresis, and isoelectrofocusing in a sucrose density gradient and assayed on in vitro-cultivated bone marrow macrophages from C57BL/6 mice. Two major MIF species, pH3-MIF with an isoelectric point of 3.0–4.3 and pH5-MIF with an isoelectric point of 4.6 to 5.2, were obtained. The similarity of murine MIF to guinea pig and human MIF is discussed.     

Antibody-induced suppression and postsuppression stimulation of complement in vitro. III. Long-term C4 suppression is actively maintained by a soluble suppressor factor (FsC4)

Previous work in our laboratory established that individual complement components can be regulated in vivo by administration of specific antibody or immunocompetent cells to newborns and in vitro by administration of specific antibody to cultured peritoneal macrophages or splenic fragments. Antibody-induced suppression of C4 was much longer lasting in cultured guinea pig splenic fragments than in cultured guinea pig peritoneal macrophages, suggesting that splenic fragments contained elements necessary for long-term suppression that were not present in the macrophage monolayers. This publication presents data in support of this concept. Antibody-treated splenic fragments from normal guinea pigs--but not from C4-deficient guinea pigs--elaborated a soluble factor (FsC4) that suppressed C4 production in previously untreated splenic fragments. FsC4 activity was most potent in splenic fragment culture supernatants at those times when intracellular and secreted C4 hemolytic activity and C4 antigen were at their lowest. C4 itself or a fragment of C4 was therefore unlikely to mediate suppression in this system. Residual anti-C4 antibody was ruled out as a mediator of FsC4 activity since it was shown by two independent methods that the amount of anti-C4 antibody carried over with the supernatant was orders of magnitude less than the amount necessary to cause suppression or to neutralize fluid phase C4 in fresh splenic fragment cultures. Preliminary data revealed that FsC4 activity may be mediated by two or more distinct molecular species or may be mediated by a single molecule that exhibits secondary size and charge heterogeneity. The identification of factors that are capable of regulating C4 suggests that, as with immunoglobulins, complement components may be regulated by complex networks of immunocompetent cells and their soluble products.     

Similarities in enhanced glucosamine incorporation by macrophages stimulated with migration inhibitory factor and the fucolectin from Lotus tetragonolobus

Fucose-binding protein (FBP), the fucolectin from Lotus tetragonolobus, was compared with migration inhibitory factor (MIF) for its ability to stimulate [¹⁴C]glucosamine incorporation into trichloroacetic acid (TCA)-insoluble material of guinea pig peritoneal exudate cells (PEC) using a microtiter assay. Both MIF and FBP inhibit macrophage migration and were shown to stimulate glucosamine incorporation in a similar dose response fashion over time. Both unpurified PEC and PEC depleted of nonadherent cells displayed significant levels of glucosamine incorporation when stimulated by MIF or FBP. Tunicamycin and 2-deoxy-d-glucose, known inhibitors of glycosylation, inhibited glucosamine incorporation by control and MIF- or FBP-stimulated PEC. These results confirm the similarities between MIF and FBP in their biological activity for macrophages using a second in vitro correlate of cell-mediated immunity and suggest involvement of enhanced glycoprotein or glycolipid biosynthesis by FBP and lymphokine-activated macrophages.     

Partial purification of antigens from eggs of Schistosoma mansoni that elicit delayed hypersensitivity

Schistosoma mansoni egg antigens that elicit delayed hypersensitivity in appropriately sensitized guinea pigs were partially characterized by using ion exchange chromatography and preparative electrophoresis. At least three skin-reactive antigens were found, one of which was purified to homogeneity, as analyzed by polyacrylamide gel electrophoresis (PAGE). This antigen was not adsorbed to CM cellulose, migrated cathodal to guinea pig albumin on electrophoresis, and was adsorbed to DEAE cellulose. A second pool of antigenic activity was obtained by adsorption to CM cellulose and subsequent elution. DEAE cellulose chromatography and preparative electrophoresis of this pool indicated the presence of more than one antigen.     

Adsorption of DNA to sand and variable degradation rates of adsorbed DNA

Adsorption and desorption of DNA and degradation of adsorbed DNA by DNase I were studied by using a flowthrough system of sand-filled glass columns. Maximum adsorption at 23 degrees C occurred within 2 h. The amounts of DNA which adsorbed to sand increased with the salt concentration (0.1 to 4 M NaCl and 1 mM to 0.2 M MgCl2), salt valency (Na+ less than Mg2+ and Ca2+), and pH (5 to 9). Maximum desorption of DNA from sand (43 to 59%) was achieved when columns were eluted with NaPO4 and NaCl for 6 h or with EDTA for 1 h. DNA did not desorb in the presence of detergents. It is concluded that adsorption proceeded by physical and chemical (Mg2+ bridging) interaction between the DNA and sand surfaces. Degradability by DNase I decreased upon adsorption of transforming DNA. When DNA adsorbed in the presence of 50 mM MgCl2, the degradation rate was higher than when it adsorbed in the presence of 20 mM MgCl2. The sensitivity to degradation of DNA adsorbed to sand at 50 mM MgCl2 decreased when the columns were eluted with 0.1 mM MgCl2 or 100 mM EDTA before application of DNase I. This indicates that at least two types of DNA-sand complexes with different accessibilities of adsorbed DNA to DNase I existed. The degradability of DNA adsorbed to minor mineral fractions (feldspar and heavy minerals) of the sand differed from that of quartz-adsorbed DNA.     

Adsorption of DNA to sand and variable degradation rates of adsorbed DNA.

Adsorption and desorption of DNA and degradation of adsorbed DNA by DNase I were studied by using a flowthrough system of sand-filled glass columns. Maximum adsorption at 23 degrees C occurred within 2 h. The amounts of DNA which adsorbed to sand increased with the salt concentration (0.1 to 4 M NaCl and 1 mM to 0.2 M MgCl2), salt valency (Na+ less than Mg2+ and Ca2+), and pH (5 to 9). Maximum desorption of DNA from sand (43 to 59%) was achieved when columns were eluted with NaPO4 and NaCl for 6 h or with EDTA for 1 h. DNA did not desorb in the presence of detergents. It is concluded that adsorption proceeded by physical and chemical (Mg2+ bridging) interaction between the DNA and sand surfaces. Degradability by DNase I decreased upon adsorption of transforming DNA. When DNA adsorbed in the presence of 50 mM MgCl2, the degradation rate was higher than when it adsorbed in the presence of 20 mM MgCl2. The sensitivity to degradation of DNA adsorbed to sand at 50 mM MgCl2 decreased when the columns were eluted with 0.1 mM MgCl2 or 100 mM EDTA before application of DNase I. This indicates that at least two types of DNA-sand complexes with different accessibilities of adsorbed DNA to DNase I existed. The degradability of DNA adsorbed to minor mineral fractions (feldspar and heavy minerals) of the sand differed from that of quartz-adsorbed DNA.     

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.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

Isolation of guinea pig macrophages bearing surface C4 by fluorescence-activated cell sorting: correlation between surface C4 antigen and C4 protein secretion

Studies of complement (C) secretion by single cells indicate that only a subset of a guinea pig macrophage population is capable of secreting the second (C2) and fourth (C4) components of C. Moreover, cell-surface bound C4 antigen is also found on a proportion of guinea pig peritoneal macrophages. The availability of methods for the isolation of macrophages bearing surface membrane C4 antigen and for the detection of the secretion of C by single cells made it possible to ascertain the relationship between these two subsets. Approximately 25% of the freshly isolated peritoneal macrophages were surface C4 antigen positive. When incubated in conditioned medium containing C4 or incubated in small volumes to increase the concentration of fluid phase C4, the proportion of macrophages bearing surface C4 increased to approximately 80%. The surface C4 antigen was adsorbed from the medium and was predominantly native C4. The proportion of peritoneal macrophages secreting functionally active C4 or C2 was approximately 45% as measured by a hemolytic plaque assay technique. The proportion of C4-secreting cells decreased to 5% after incubation in conditioned medium containing preformed C4, whereas the proportion of C2-producing macrophages was unchanged, i.e., it remained at about 45%. The removal of secreted C4 with F(ab')2 anti-C4 or effectively decreasing C4 concentration by increasing the volume of the culture medium abrogated the decrease in the proportion of C4-secreting cells. Conditioned medium derived from cells genetically deficient in C4 had no effect on the proportion of C4- or C2-producing macrophages. The macrophage population bearing surface membrane C4, isolated by fluorescence-activated cell sorting, contained more than 90% of the C4-producing cells. Cells producing C2 were distributed equally in both subpopulations. Maintenance of the surface C4-positive, C4-producing cells in culture for 12 hr resulted in a decrease in the proportion of C4-secreting cells. Conversely, isolated macrophages initially surface C4 negative and not producing C4, developed the capacity to produce C4 in culture. C4 production in the isolated surface C4-negative population was inhibited by incubation in medium containing preformed C4. These results suggest the presence of a negative feedback effect on C4 secretion that is mediated by extracellular C4.(ABSTRACT TRUNCATED AT 400 WORDS)