Monocyte activating factor in sarcoidosis. II. Secretion of the factor from monocytes

The normal human monocytes pretreated with the monocyte activating factor found in sarcoidosis sera were shown to secrete a factor which induced normal human monocytes to spread as well as to increase their cell size. Phagocytosis and glucose consumption of normal human monocytes were also increased by this secondarily obtained factor. Its molecular weight was about 70,000. These results indicate that this secondary factor may be the same substance as the monocyte activating factor found in sarcoidosis sera. The normal human monocytes pretreated with the monocyte activating factor were also shown to liberate a factor which generated macrophage migration inhibitory factor in cooperation with normal human sera.     

An inhibitory factor against monocyte spreading in the sera of patients with systemic lupus erythematosus

The effect of the sera of patients with systemic lupus erythematosus (SLE) on monocyte function was studied using cell spreading as an indicator. Monocyte spreading induced by exogenous stimuli was shown to be inhibited by SLE sera. Gel filtration of SLE sera on Sephadex G-200 revealed that the factor responsible for this inhibition had a molecular weight of about 50,000. Pretreatment of monocytes with the inhibitory factor led to suppression of cell spreading induced by subsequent stimulation, but this hyporeactivity was reversible. Spreading of monocytes was rapidly aborted by the addition of this inhibitory factor. Thus, the inhibitory factor appeared to affect monocyte itself, but its effect seemed to be transient.     

Studies on migration inhibitory factors of non-lymphoid origin

A large number of mouse fibrosarcoma and adult guinea pig fibroblast cultures were examined for their ability to produce migration inhibitory activity. In most cases culture supernatants were found to inhibit macrophage migration, in a dose-dependent manner. Toxicity of the tested material could be excluded by: a) experiments using colchicine as a stimulator of macrophage migration and, b) examination of the effect of test materials on macrophage monolayer cultures. Additionally, migration inhibitory activity was found in fibroblast, but not fibrosarcoma frozen and thawed extracts. Furthermore, incubation of cells with puromycin could only inhibit production by fibrosarcoma, thus suggesting that the fibroblast activity was due to preformed cellular constituents. Fractionation of concentrated culture supernatants by Sephadex G-200 gel filtration showed that the activity derived from fibrosarcoma cells could be eluted in a narrow molecular weight fraction (18,000-22,500), whereas the fibroblast activity was heterogenously distributed over a wide range. Migration inhibitory activity in fibroblast extracts was mainly associated with higher molecular weight material. Differences could be demonstrated between these activities and lymphocyte migration inhibitory factor, including inhibition by methyl-pentoses and the presence of macrophage aggregating activity.     

Colony stimulating factor (CSF) activity in human and murine sera.

Substances with CSF activity were studied in human and murine sera. Gel filtration of the sera on Sephadex G-200 showed that fractions with molecular weight of 14,000 and 6000 D were active in the CSF test. When mice were stimulated with endotoxin, CSF activity of both fractions was twice as high as in control animals, and CSF activity was found also in a protein peak with the molecular weight of 300,000 D. The fractions with CSF activity were sensitive to pepsin.     

Production of macrophage migration inhibitory factor in rabbits with experimental arthritis

Cultures of synovial tissue from rabbits with an antigen-induced arthritis were tested for production of macrophage migration inhibitory factor to investigate the possible role of cellular immunity in this experimental model. Culture supernatants of ten specimens of normal rabbit synovium and six specimens from joints injected with saline did not inhibit macrophage migration; and in thirteen experiments with synovium from an arthritis produced by injection of urate crystals only one showed significant inhibitory activity on macrophages. In contrast nineteen of twenty-five antigen challenged joints produced migration inhibitory factor detectable in the culture supernatants. Inhibition ranged from 20 to 87 per cent with a mean of 31·7±4·3 per cent. The migration inhibitory factor in these fluids was non-dialysable and was eluted with an albumin marker from Sephadex G-200. The finding that a macrophage migration inhibitory agent is produced in the course of antigen-induced synovitis is consistent with the postulated role of cellular immunity in the synovial lesion.     

Studies on the inhibition of macrophage migration induced by soluble antigen-antibody complexes.

Mixtures of serum of Freund's complete adjuvant (FCA) immunized guinea-pigs and tuberculin PPD consistently inhibited the in vitro migration of peritoneal exudate cells (PEC) of normal guinea-pigs. It is shown that this inhibitory effect is due to a soluble complex between an IgG2 antibody and PPD. By separation of PPD on Sephadex G-200 two peaks were obtained, corresponding respectively to molecular weights of at least 800,000 and 25,000, separated by a plateau. Material derived from both peaks and from the plateau was able to form inhibitory complexes with anti-PPD IgG2. When a mixture of small molecular weight PPD and anti-PPD IgG2 was fractionated on Sephadex G-200, the inhibitory activity was recovered in the void region only. The detection of both IgG2 and PPD in the latter was taken as evidence for the presence of a high molecular weight antigen-antibody complex. When the mechanism of complex-induced inhibition of migration was examined it was found that: (1) complexes act directly on macrophages present in the peritoneal exudate; (2) removal of the Fc fragment of IgG2 by pepsin abolishes its ability to form migration inhibitory complexes; (3) passive sensitization of macrophages with anti-PPD IgG2, followed by exposure to PPD does not result in inhibition of migration; (4) in order to obtain migration inhibition, the complexes must be present during the entire migration period. A 2-hr pulse with complexes does not induce permanent inhibition; (5) the migration inhibitory activity of antigen--antibody complexes can be abolished by certain concentrations of puromycin and aminophylline.     

Antigen and mitogen induced production of macrophage migration inhibitory factor in the mouse.

Spleen cells of C57B1/6J mice immunized with complete Freund's adjuvant produced macrophage migration inhibitory factor (MIF) when incubated in vitro with tuberculin purified protein derivative (PPD). For optimal MIF production spleen cells were cultured for 48 h in a serum-free medium, at a concentration of 2 x 10(7) cells/ml. MIF was assayed in a xenogenic system, using oil-induced guinea pig peritoneal exudate cells as targets. MIF synthesis could also be induced by pulsing spleen cells for 2 h with concanavalin A, phytohemagglutinin, pokeweed mitogen or lipopolysaccharide, followed by culture in plain medium. No MIF secretion was induced by incubation of spleen cells with anti-theta or rabbit anti-mouse IgG sera. Cells producing MIF in response to PPD were characterized as B cells by virtue of being insensitive to anti-theta serum and complement, by being retained on nylon wool, glass bead and anti-Ig colums and by the presence of Fc receptors. PPD-stimulated T cells did not produce MIF. PPD-induced mouse spleen cell MIF demonstrated a moderate loss of activity by heating at 56 and 80 degrees C and was completely inactivated after digestion with chymotrypsin. By fractionation on Sephadex G-200, migration inhibitory activity was recovered in a molecular range of 100,000-12,400 daltons.     

A phagocytosis-enhancing factor in human plasma.

A phagocytosis-enhancing factor (PEF) with the capacity to stimulate the ingestion of sensitized sheep erythrocytes by human polymorphonuclear and mononuclear leucocytes has been isolated from human plasma by chromatography on DEAE-cellulose and filtration on Sephadex G-150 and Sephadex G-100. PEF is a protein of approximately 70,000 molecular weight which is susceptible to inactivation by heating at 60 degrees or by tryptic digestion. PEF promotes phagocytosis of erythrocytes sensitized with intact 7S antibody or bearing the C3b complement fragment, but not of unsensitized erythrocytes or erythrocytes sensitized with 19S antibody. The specificity of PEF interaction with target erythrocytes and the persistence of its stimulatory effect after the target cells are washed suggest that it promotes phagocytosis by an action on the erythrocytes.     

Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response.     

A sensitive technique for measuring specific macrophage aggregation: a comparison with macrophage migration inhibition, for the detection of lymphokine activity.

A new quantitative method for the measurement of macrophage aggregation, induced by lymphokine preparations, is described. This involves the continuous measurement of the light absorbance of stirred macrophage suspensions. The results have been compared with those obtained by measuring macrophage migration inhibition activity for the detection of lymphokine activity. Separation of crude lymphocyte culture supernatants by Sephadex G-200 shows that the material with aggregating activity is heterogeneous. The greater amount of this activity is not separated from macrophage migration inhibition activity, and has a molecular weight of 35,000--70,000. A comparison has been made with other published methods for measuring macrophage aggregation.     

Studies on the physico-chemical characteristics of polymorph migration stimulator.

Polymorph migration stimulator is a supernatant factor produced by the interaction between glucocorticosteroids and human blood monocytes in culture. Studies on the physical characteristics of this factor show that it is soluble and stable at high and low temperatures. Its activity is reduced by acid and alkali treatment and destroyed by the proteolytic enzyme protease. Experiments involving dialysis, ultrafiltration and Sephadex G-100 gell filtration indicate that the molecular weight of the polymorph migration stimulator is between 12,000 and 15,000. It is suggested that this factor may mediate the anti-inflammatory effects of glucocorticosteroids on phagocytic cells.     

The mediation of tissue eosinophilia in hypersensitivity reactions. II. Separation of a delayed eosinophil chemotactic factor from macrophage chemotactic factors.

In anaphylactic cutaneous lesions induced by DNP-ascaris extract in the guinea-pig, the time-course of delayed tissue eosinophilia was found to parallel that of the macrophage reaction, reaching its peak in 24 h. Macrophages could be differentiated from lymphocytes by the numerous lysosomal granules which stained for acid phosphatase. Extracts from such skin lesions contained a delayed eosinophil chemotactic factor and two different macrophage chemotactic factors. Most of the delayed eosinophil chemotactic factor was separated from the two macrophage chemotactic factors by gel filtration on Sephadex G-100 and Sephadex G-200 in that order. The eosinophil chemotactic factor after re-chromatography on Sephadex G-I99 showed no or little chemotactic activity for macrophages.     

Enhancement of eosinophil leukocyte chemotaxis by a factor in normal human serum (SEF).

An activity in normal human serum is described which enhances the migration of eosinophils towards neutrophil-derived eosinophil chemotactic factor (ECF). The serum-enhancing factor (SEF), in contrast to the inhibitors in serum, is expressed primarily during weak chemotaxis, acts in a cell-directed fashion, increases the chemokinesis of eosinophils and, in the presence of ECF, enhances chemotaxis in a synergistic fashion. SEF is found in sera of several mammalian species, affects human and guinea pig eosinophils, and it is heat-labile (56 degrees C). On Sephadex column chromatography, SEF has a molecular weight of approximately 800,000 daltons. In some sera, a second activity with a molecular weight of approximately 200,000 daltons is eluted. SEF does not enhance eosinophil migration once the cells have been deactivated in vitro by their chemotactic factor. Because of its wide distribution, SEF may play an important modulating role both in in vitro assay systems and in vivo.     

Antihaemophilic factor (factor VIII) from human granulocytes.

Factor VIII has been isolated and purified from human granulocytes using chromatography on Sephadex G-200 AND DEAE-Sephadex A-50. The final preparation was purified 60-fold. Some properties of this partly purified factor VIII were compared with the human plasma AHF preparation. Optimum pH activity and the highest activity after incubation at different pH values was found in buffer at pH 7.0. The preparation was thermolabile and had a molecular weight of about 214,000. Absorption curves indicated that it is a protein exhibiting four fractions in polyacrylamide gel disk electrophoresis. AHF preparations from plasma and granulocytes were almost identical in the characteristics tested.     

Characterization of a factor in leprosy serum that inhibits the growth of mitogen-stimulated normal human lymphocytes.

A factor that inhibits the growth of mitogen-stimulated lymphocytes from normal donors has been detected in the sera of patients with chronic leprosy. The inhibitory activity was detected with similar frequency in patients with tuberculoid or lepromatous leprosy, although higher levels of activity were detected in the latter. The factor reduced the growth in volume of the lymphocytes in the first 24 hr after stimulation, the synthesis of RNA during the first 3 days of culture and the replication of DNA in 72-hr cultures. All the inhibitory activity co-purified with IgG on gel filtration, ammonium sulphate fractionation and ion exchange chromatography. The activity was stable to heating at 56 degrees but labile at 100 degrees and was absorbed from serum or from purified IgG preparations by staphylococcal protein A. On gel filtration of the sera on Sephadex G-200, none of the activity appeared in the void volume, indicating that it is not due to immune complexes. We conclude that the activity is due to an IgG antibody and suggest that it is an autoantibody since the sera inhibited the growth of all donor lymphocytes tested.     

The Fractionation of Antigen-Dependent Macrophage Migration Inhibition and Macrophage Activation Factors from Lymph Draining a Tuberculin Reaction

Efferent lymph, taken from BCG-sensitized sheep 6 to 20 hr after inaction of PPD, was fractionated by chromatography on Sephadex G-200 and on Sepharoseconcanavalin A and by rechromatography on Sephadex G-200 The fractions were examined for their ability to inhibit the migration of normal guinea pig peritoneal exudate cells (MIF assay) and to stimulate the uptake of colloidal gold by these cells (GUS assay). Antigen-dependent MIF activity and GUS activity eluted from Sephadex G 200 in the albumin fraction. No corresponding activities were found in this region when control lymph was fractionated in an identical manner. After chromatography on Sepharoseconcanavalin A, antigen-dependent MIF activity was associated with a glycoprotein fraction containing IgG. After rechromatography on Sephadex G-200, antigen-dependent MIF and GUS activities eluted at a position corresponding with that of proteins of 60,000 to 70,000 molecular weight, and these fractions contained less than 2.5 μg/ml IgG. Soluble mediators produced in vivo by sheep with a delayed hypersensitivity response to PPD possessed antigen dependent MIF activity. The antigen-dependent macrophage-inhibiting and macro phage-activating factors were closely associated in fractions that did not appear to contain significant amounts of IgG.     

Characterization of a T-lymphocyte inhibitor in the serum of tumour-bearing mice.

Sera from mice with large tumours from a variety of tissue types and sources have been shown to contain substances capable of suppressing the proliferative response of normal mouse lymphocytes to concanavalin A (Con A), bacterial endotoxin (LPS) and allogeneic cells. The present paper deals with studies on the nature of these inhibitory materials using mainly a methylcholanthrene-induced rhabdomyosarcoma in DBA/2J mice. It was found that a material responsible for inhibition of the Con A response eluted with immunoglobulins on Sephadex G-150 and eluted with monomeric immunoglobulin on Sephadex G-200. The component of tumour-bearer serum responsible for the suppression of the LPS response of normal lymphocytes eluted from Sephadex G-150 with the alpha and beta globulins and albumin (molecular weight less than 150,000). The immunoglobulin-containing serum fraction from tumour-bearing animals inhibited the mixed lymphocyte response, Con A response, and specific immune response to purified protein derivative (PPD) in allogeneic cell systems. It also inhibited the in vitro primary response of mouse cells to sheep red blood cells, and, to a lesser extent, the response to a T cell-independent antigen (DNP-dextran). The inhibitory activity continued to elute with monomeric IgG on Sephadex G-200 when columns were run in 1640 medium and adjusted to pH 2-5, indicating that an acid dissociable complex was not responsible for inhibitory activity. Inhibitor activity could be removed by absorption on immuno-adsorbents containing goat anti-mouse immuno-globulin, and could be recovered by acid elution from the absorbent. Inhibitor activity was not removed by immunoadsorption on columns prepared with antisera to chicken immunoglobulin.     

Functional role of interleukin 1 in periodontal disease: induction of interleukin 1 production by Bacteroides gingivalis lipopolysaccharide in peritoneal macrophages from C3H/HeN and C3H/HeJ mice.

Hot phenol-water-extracted lipopolysaccharide (LPS) from Bacteroides gingivalis 381 was purified by Sephadex G-100 chromatography with Tris buffer supplemented with sodium deoxycholate and EDTA (B-LPS). In the present study, B-LPS was examined for its ability to induce interleukin 1 (IL-1) production, a mitogenic response, and macrophage activation in LPS high-responder C3H/HeN and low-responder C3H/HeJ mice. A significant increase in IL-1 production was observed in C3H/HeN and C3H/HeJ peritoneal macrophages treated with various doses (1.0 to 50 micrograms/ml) of B-LPS. IL-1 production by C3H/HeN macrophages treated with B-LPS (10 micrograms/ml) was about seven times greater than that by C3H/HeJ macrophages. However, the IL-1 production induced by B-LPS (10 micrograms/ml) in C3H/HeN macrophages was four times lower compared with that induced by Escherichia coli O111 B4 LPS. Also, a significant increase in IL-1 production was found in human monocytes stimulated with B-LPS. That B-LPS-induced IL-1 exhibits some molecular weight heterogeneity was indicated from Sephadex G-75 gel filtration profiles. A significant, high mitogenic response by whole spleen cells with 1 X 10(5) to 5 X 10(4) cells of either mouse strain per well treated with B-LPS (10 to 50 micrograms/ml) was observed. However, the response of C3H/HeJ mice was less than that of the C3H/HeN strain. Also, glucose consumption assays indicated that enhanced macrophage activation occurred in C3H/HeN but not in C3H/HeJ mice treated with B-LPS. In light of recent studies showing that IL-1 stimulates bone resorption in a mouse calvaria system and collagenase production in fibroblasts, we suggest that B-LPS-induced IL-1 may play a significant role in the pathogenesis of adult periodontal disease.     

Bacterial-lipopolysaccharide-induced release of lactoferrin from human polymorphonuclear leukocytes: role of monocyte-derived tumor necrosis factor alpha.

We have examined the role played by human peripheral blood monocytes in mediating responses of human polymorphonuclear leukocytes (PMN) to bacterial lipopolysaccharide (LPS) in vitro. When incubated with Salmonella typhimurium LPS at 37 degrees C, human PMN suspended in serum-free buffer released the specific granule constituent lactoferrin into the surrounding medium. Release of lactoferrin from PMN varied with the concentration of LPS (1 to 1,000 ng/ml) as well as with the duration of incubation (2 to 60 min) and was not accompanied by significant release of the cytoplasmic enzyme lactate dehydrogenase. LPS-induced release of lactoferrin from PMN was augmented significantly when cell suspensions were supplemented with additional monocytes and lymphocytes. Only monocytes, however, secreted significant amounts of lactoferrin-releasing activity (in a time- and concentration-dependent manner) when incubated separately with LPS. Lactoferrin-releasing activity was heat (80 degrees C for 15 min) labile, eluted after chromatography on Sephadex G-100 with an apparent molecular weight of approximately 60,000, and was inhibited by antibodies to tumor necrosis factor alpha. Thus, LPS-induced noncytotoxic release of lactoferrin from human PMN suspended in serum-free buffer is mediated, at least in part, by tumor necrosis factor alpha derived from contaminating monocytes.     

Elution patterns of rubella IgM, IgA, and IgG antibodies from a dextran and an agarose gel.

The elution pattern of serum proteins and the distribution of rubella HAI activity in 95 sera from 65 cases were determined after gel filtration with (a) Sephadex G-200 and (b) Bio-Gel A-5M. Rubella HAI antibody in peak 1 after Sephadex G-200 fractionation of early convalescent sera consists of both IgM and high molecular weight IgA. However, these two classes of antibody can be distinguished by gel filtration with Bio-Gel A-5M. Bearing these differences in mind, the results of fractionation with the two gels correlate very well although the use of Bio-Gel A-5M gives a slightly less sensitive technique for the diagnosis of recent infection.