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.     

Stimulation of DNA synthesis in cultured mouse peritoneal macrophages after fusion injection of macrophage growth factor

Macrophage growth factor (MGF), when injected into cultivated mouse peritoneal macrophages by Sendai virus-fusion with loaded human erythrocyte ghosts, induced macrophage DNA synthesis within 40 h. DNA synthesis was observed in approx. 10% of the injected cells. Fusion products between macrophages and red cell ghosts were identified by co-injection of ¹²⁵I-labelled human serum albumin. These studies suggest that the mechanism of the physiological effect of MGF may depend on the endocytosis of at least part of the MGF molecule.     

Identification of a ligand for the c-kit proto-oncogene.

We report the purification and N-terminal amino acid sequence of a novel mast cell growth factor, termed MGF, from the supernatants of a murine stromal cell line. A panel of interleukin 3-dependent cell lines were screened for responsiveness to partially purified MGF in [3H]thymidine incorporation assays; proliferative stimulation of these cells in response to MGF correlated with expression of mRNA for the c-kit protooncogene. MGF was shown to be a ligand for c-kit by cross-linking 125I-labeled MGF to c-kit-expressing cells with subsequent immunoprecipitation of the complex with antiserum specific for the C-terminus of c-kit. This establishes MGF as a ligand for the c-kit protein.     

Regulation of DNA synthesis in mouse macrophages. II. Studies on mechanisms of action of the macrophage growth factor

When mouse macrophages are incubated with medium conditioned by mouse fibroblasts, they are induced to synthesize DNA and divide. This phenomenon is triggered by a macrophage growing factor (MGF) released by the fibroblasts. The presence of a serum cofactor is essential to the activity of the MGF; this cofactor can be removed by dialysis and seems unrelated to the “growth-promoting substances” normally present in serum. The kinetics of DNA synthesis in macrophages stimulated by conditioned medium (CM) is characterized by a lag phase of 24–48 h and a peak synthesis at 4–5 days, followed by a rapid decrease. This decrease is caused by depletion of the MGF from the CM by the growing macrophages.     

Induction of transforming growth factor-alpha in activated human alveolar macrophages

The early monocyte infiltration observed in normal wound repair and in a number of pathologic processes precedes the epithelial and connective tissue proliferative responses, suggesting that the monocyte/macrophage may be an important source of growth factors for these tissues. In culture, activated macrophages secrete growth factors active on fibroblasts, smooth muscle, endothelium, and epithelium. This report demonstrates that activated human alveolar macrophages express the gene for transforming growth factor-alpha (TGF-alpha) in an inducible manner and secrete a factor into the culture medium that is functionally and immunologically identical to TGF-alpha. Two different molecular species of TGF-alpha activity (approximately 8,500-12,000 and 28,500 daltons) are identified in macrophage-conditioned medium. These observations establish the macrophage as a diploid human cell capable of synthesizing and secreting TGF-alpha. The activated macrophage therefore represents a cellular source of a mitogenic factor that is potentially important in epithelial proliferation and repair.     

Apoptosis of sea bass (Dicentrarchus labrax L.) neutrophils and macrophages induced by experimental infection with Photobacterium damselae subsp. piscicida

The infection of sea bass (Dicentrarchus labrax L.) by intraperitoneal (i.p.) injection of the agent of fish pasteurellosis Photobacterium damselae subsp. piscicida resulted in the apoptosis of peritoneal neutrophils and macrophages. All the eight virulent and none of the two non-virulent strains tested exhibited apoptogenic activity. A secreted bacterial protein(s) is a likely candidate as the factor(s) responsible for this activity, since no apoptosis was induced by i.p. injected UV-killed virulent strains and the virulent culture supernatants exhibited a thermo-labile apoptogenic activity identical to that of live bacteria. The apoptotic process was characterized by the occurrence of DNA fragmentation detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining and DNA electrophoresis, and of typical ultrastructural alterations namely cell shrinkage, chromatin condensation, nuclear fragmentation and production of blebs with shedding of apoptotic bodies. In the apoptotic process induced by lethal doses of virulent bacteria or culture supernatants both peritoneal macrophages and neutrophils were extensively affected, the majority of these cells being apoptotic and reaching values around 10(7)per peritoneal cavity for each cell type at 24h post-injection. Moreover, the number of non-apoptotic macrophages was always below the initial number in the resting peritoneal cavity. Since macrophages are key cells in the elimination of both bacteria and apoptotic moribund cells and apoptotic bodies, the induction by Ph. damselae subsp. piscicida of simultaneous macrophage and neutrophil apoptosis results, on the one hand, in the destruction of the two phagocytic cell types involved in the restriction of multiplication of the bacteria and, on the other hand, in the uncontrolled progression of the apoptotic process towards secondary necrosis and eventual lysis of high numbers of moribund neutrophils and of neutrophilic apoptotic bodies, with the consequent extensive release of their highly cytotoxic components. Abundant apoptotic cells were also seen in sections of head-kidney from fish dying from experimental pasteurellosis. In contrast, no apoptosis was seen in vitro after the treatment with virulent culture supernatants of sea bass head-kidney macrophage cultures or after the treatment ex vivo of peritoneal exudate leukocytes with virulent bacteria or culture supernatants. The apoptotic process described here appears as a novel and very powerful microbial pathogenic strategy.     

Lymphokine production by human T cell hybridomas

Human T cell hybridomas were generated by hybridization of SH9 cells, the 6-thioguanine-resistant variant of human T lymphoma Hut102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. The hybrid nature of the established cell lines was documented by the difference in D14S1 restriction fragment length polymorphism between SH9 and the hybridoma cell DNA, and by the expression of OKT11 antigen on hybrid cells. T cell growth factor, macrophage growth factor (MGF), and interferon (IFN) activities have been demonstrated in the supernatants of different hybrid cultures, but not in SH9 cell cultures. Substantial quantities of MGF were secreted by several hybrids including the L23 line. MGF activity was dose-dependent, heat-labile, and synergistic with indomethacin. High titers of IFN activity were found in the cultures of hybridoma L415 and its subclones. Neutralization with specific antisera showed the IFN synthesized by L415 clones was immune interferon (IFN-gamma). Like the parental SH9 line, all of the hybridomas producing these lymphokines exhibited a cell surface phenotype typical for helper T cells. The hybridoma system therefore shows potential for the study of various lymphokines produced by human helper T lymphocytes.     

Glucocorticoids and prostaglandins inhibit the induction of macrophage DNA synthesis by macrophage growth factor and phorbol ester

The tumor-promoting phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), stimulates starch-elicited mouse peritoneal macrophages to undergo DNA synthesis in vitro, apparently without the generation of an endogenous macrophage growth factor (MGF). No evidence was found for any synergistic interaction between TPA and exogenous colony stimulating factors (CSFs) for macrophage DNA synthesis. Low concentrations of glucocorticoids and also prostaglandins E1 and E2 suppress both the CSF-1-stimulated and the TPA-stimulated macrophage DNA synthesis; these same drugs inhibit the CSF-1-mediated and TPA-mediated enhancement of macrophage plasminogen activator (PA) activity. Thus glucocorticoids and prostaglandins E1 and E2 oppose the action of growth factors and the tumor promoter on macrophage and precursor cell function.     

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.     

Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells

A major function of macrophages is to engulf apoptotic cells to prevent them from releasing noxious materials as they die. Milk fat globule-EGF-factor 8 (MFG-E8) is a glycoprotein secreted by activated macrophages that works as a bridge between apoptotic cells and phagocytes by specifically recognizing phosphatidylserine exposed on apoptotic cells. In this study, we found that developmental endothelial locus-1 (Del-1), originally identified as an embryonic endothelial cell protein that binds alphavbeta3 integrin, is structurally and functionally homologous to MFG-E8. That is, both consist of a signal sequence, two epidermal growth factor domains and two factor VIII-homologous domains (C1 and C2). Del-1 bound to the apoptotic cells by recognizing phosphatidylserine via the factor VIII-homologous domains with an affinity similar to that of MFG-E8. The phagocytic activity of NIH 3T3 cells against apoptotic cells was enhanced by Del-1 through an interaction between the epidermal growth factor domain in Del-1 and alphavbeta3 integrin expressed in the NIH 3T3 cells. Screening of primary macrophages and macrophage cell lines for the expression of MFG-E8 and Del-1 indicated that MFG-E8 and Del-1 are expressed in different sets of macrophages. These results suggest the existence of macrophage subsets that use MFG-E8 or Del-1 differently to engulf apoptotic cells.     

Role of granulocyte/macrophage colony-stimulating factor in the regulation of murine alveolar macrophage proliferation and differentiation

Granulocyte/macrophage (GM)-CSF is one of the hemopoietic growth factors that stimulates neutrophilic granulocyte and macrophage production by bone marrow progenitor cells. In this study, the effect of GM-CSF on the growth and differentiation of murine pulmonary alveolar macrophages (PAM) was investigated. In the presence of GM-CSF, normal murine PAM were induced to proliferate and develop into macrophage colonies with a dose-response curve similar to that of bone marrow GM colony-forming cells. PAM also responded to CSF-1, a lineage-restricted growth factor, but required much higher doses of CSF-1 and a longer incubation time for optimal colony formation. The proliferative response of PAM to CSF-1, however, was greatly enhanced by the concurrent addition of low doses of GM-CSF. In contrast, low doses of CSF-1 failed to potentiate the proliferative response of PAM to GM-CSF. Macrophages derived from GM-CSF cultures were rounder and less stretched and possessed less FcR-mediated phagocytic activity than cells produced in CSF-1 cultures. A study with hydrocortisone-induced monocytopenia showed that nearly one half of lung macrophages may be sustained by local proliferation of PAM without the continuous migration of blood monocytes. This study suggests that GM-CSF may play a major role in the production of PAM by two modes of action, 1) direct stimulation of cell proliferation and 2) enhancement of their responsiveness to CSF-1, thereby producing more mature and functionally competent macrophages.     

Characterization of the growth-inhibitory and apoptosis-inducing activities of a triterpene saponin,securioside B against BAC1.2F5 macrophages

BACKGROUND: Since the growth state of macrophages in local pathological sites is considered a factor that regulates the processes of many disease, such as tumors, inflammation, and atherosclerosis, the substances that regulate macrophage growth or survival may be useful for disease control. We previously reported that securiosides A and B, novel triterpene saponins, exerted macrophage-oriented cytotoxicity in the presence of a L-cell-conditioned medium containing macrophage colony-stimulating factor (M-CSF), while the compounds did not exhibit an effect on macrophages in the absence of the growth-stimulating factors. AIM: This study was undertaken to characterize the growth-inhibitory and the apoptosis-inducing activities of securioside B, focusing on the effects of the macrophage-growth factor(s), and to examine the implication of a mitochondria pathway in apoptosis induction. METHODS: The effect of securioside B on a murine macrophage cell line (BAC1.2F5) was examined by MTT assay and lactose dehydrogenase release assay in the presence of L-cell-conditioned medium, M-CSF, or granulocyte-macrophage CSF (GM-CSF). RESULT: Securioside B inhibited the growth of the cells stimulated by recombinant M-CSF or GM-CSF, but it scarcely induced cytolysis of the cells under the same conditions. Moreover, securioside B did not induce cell death when the compound only was added to the cells. On the other hand, the compound extensively induced apoptotic cell death in the presence of L-cell-conditioned medium, suggesting that apoptosis induction by securioside B requires the additional factor(s) present in L-cell-conditioned medium. Securioside B plus L-cell-conditioned medium induced the activation of caspase-3 and caspase-9, but not caspase-8. In addition, cytochrome c release from the mitochondria into the cytosol, and disrupted mitochondria membrane potential, was also observed in the apoptotic BAC1.2F5 cells. CONCLUSION: These data suggest that securioside B has growth-inhibitory activity against growth factor-stimulated macrophages, and that it induces apoptotic macrophage death through the activation of a mitochondrial pathway in the presence of L-cell-conditioned medium.     

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro

 Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i. p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage- colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i. p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth. Received: 11 April 1996 / Accepted: 21 May 1996     

Effects of serum fractions on the growth of mononuclear phagocytes

The effects of serum fractions on the growth kinetics and colony formation of mononuclear phagocytes derived from mouse bone marrow, blood, and peritoneal cavity were investigated. Peritoneal exudate macrophages and blood monocytes required a factor(s) found to reside in the nondialyzable serum fraction (molecular weight > 12,000) to survive, a small molecular weight (< 307) factor(s) with growth-stimulatory activity (GSA) contained in the dialyzable serum fraction, and the macrophage growth factor (MGF) for proliferation and colony formation. Fetuin, a major protein of fetal serum, was able to substitute the non-dialyzable serum fraction. Macrophages cultured in medium containing MGF and the nondialyzable serum fraction for 6 days could be restored to full growth following the addition of the dialyzable serum fraction. In contrast, bone marrow mononuclear phagocytes cultured in the absence of the dialyzable serum fraction were capable of proliferating, though at a slower rate, and forming colonies. In addition, neither insulin nor hydrocortisone was capable of replacing the serum-dialyzable GSA nor able to enhance colony formation.     

Cytokinetic analysis of the expanding Kupffer-cell population in rat liver

Zymosan stimulation in rats provides a useful model for studying the expansion of the Kupffer-cell population in liver, which represents the major population of tissue macrophages. This study, using tritiated-thymidine-labelling experiments, demonstrates that during population expansion at least 90% of the resident macrophages (Kupffer cells) develop proliferative activity. The mean duration of the cell cycle is estimated to be 52 hr, with an S phase of 7 hr. We have calculated that about 75% of population expansion results from local Kupffer-cell replication, whereas the remaining growth results from extrahepatic recruitment of macrophage precursors. These findings conflict with a concept of the mononuclear phagocyte system, which states that resident macrophages are (monocyte-derived) non-dividing end-cells.     

Identification of IL-6 as a T cell-derived factor that enhances the proliferative response of thymocytes to IL-4 and phorbol myristate acetate

Thymocytes undergo a vigorous proliferative response when stimulated with a combination of IL-4 and PMA. We have found that conA-induced supernatants from a number of Th cell clones could enhance the level of IL-4/PMA-induced proliferation of unseparated thymocytes 0.5- to 2-fold and of peanut agglutinin-positive thymocytes 2- to 10-fold. These supernatants did not contain IL-2 or IFN-gamma, and the enhancing activity could be chromatographically separated from IL-3, -4, -5, and granulocyte/macrophage CSF. The possibility that the thymocyte enhancement factor contained in these supernatants was IL-6 was suggested when murine rIL-6 was found to have similar activity. Further evidence for the identity of these two factors was obtained when an IL-6 assay, based on plasmacytoma growth, was used to test column fractions showing thymocyte enhancement. All fractions active in the thymocyte enhancement assay also had activity in the plasmacytoma growth assay. These observations suggest that the thymocyte-stimulating activity present in the T cell supernatants was due to IL-6.     

Induction of tumouricidal macrophages by MTP-PE: its enhancement by a factor produced spontaneously by tumour cells

Tumouricidal activity of rat alveolar macrophages is induced by MTP-PE in vitro. This tumouricidal activity is enhanced by a factor (tumour cell derived immunostimulating factor, TCIF) contained in tumour cell culture supernatants. TCIF is not species specific, since culture supernatants of rat MADB-200 as well as mouse B16 or Meth A tumour cells showed similar effects on rat alveolar macrophages. TCIF is not produced in cultures of normal cells, e.g. rat embryo cells. TCIF produced by MADB-200 tumour cells is relatively heat-stable and dialyzable. It is destroyed by treatment at pH 2 for 24 hrs. These results suggest that TCIF can participate in macrophage activation and could be of potential therapeutic value.     

Reversal of macrophage-augmented MLR reactivity by tumor-induced splenic suppressor T cells and their soluble factor(s)

Augmenting concentrations of macrophages or their supernatants failed to reverse T-cell hyporeactivity in tumor-bearing mice (TBM). Serial passaging over nylon wool columns depleted TBM spleen cells of a mildly adherent tumor-induced suppressor cell and restored mixed lymphocyte reaction (MLR) reactivity to the purified TBM T-cell population. The tumor-induced suppressor cell was extensively plated to remove macrophages and characterized as a T cell by its anti-Thy 1 serum sensitivity. This suppressor T cell, when added to normal T cells, abrogated all enhancing effects caused by addition of macrophages. Suppressor T-cell inhibition was non-contact dependent, since suppressor T-cell supernatants inhibited MLR activity in T cells treated with enhancing concentrations of macrophage supernatants. Thus it appears that tumor-induced T-cell debilitation is a reversible phenomenon, mediated not by macrophages but by soluble factor(s) from a nonphagocytic, mildly adherent, suppressor T cell.