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Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that is responsible for almost 1.5 million deaths per year. Sensing of mycobacteria by the host's immune system relies on different families of receptors present on innate immune cells. Amongst them, several members of the TLR family are involved in the activation of immune cells by mycobacteria, yet the in vivo contribution of individual TLRs to the protective immune response remains controversial. On the contrary, MyD88, the adaptor molecule for most TLRs, plays a non-redundant role in the protection against tuberculosis and mice with a complete germline deletion of MyD88 succumb very early to infection. MyD88 is expressed in both immune and non-immune cells, but it is not clear whether control of mycobacteria requires ubiquitous or cell-type specific MyD88 expression. Therefore, using novel conditional switch-on mouse models, we aimed to investigate the importance of MyD88 signalling in DCs and macrophages for the induction of protective effector mechanisms against mycobacterial infection. We conclude that specific reactivation of MyD88 signalling in CD11c- or lysozyme M-expressing myeloid cells during Mycobacterium bovis Bacille Calmette-Guerin infection is sufficient to restore systemic and local inflammatory cytokine production and to control pathogen burden., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Macrophage function is an important factor for resistance to infection and anti-oxidative vitamins and minerals can affect how macrophages function in fish. We report the in vivo effect of adding singular or combined vitamins (A, C, and E) and/or minerals (Se, Zn, Cu, Mn, and Fe) in diets on the immune system of tilapia (Oreochromis hybrids) peripheral blood monocyte-derived, anterior kidney-derived, and spleen-derived macrophages. An optimal dose of vitamins and/or minerals in diets increased macrophage proliferation and protective activity, maintained macrophage viability, increased body weight and length, and increased lysozyme activity, however, at improper doses and combinations of vitamins or minerals a decrease was observed. Furthermore, vitamins and/or minerals at any doses and combinations in diets decreased superoxide and nitric oxide production. Therefore, appropriate doses and combinations of vitamins and/or minerals in diets may increase tilapia macrophages immunity.

Atlantic salmon head kidney macrophages grown in the presence of particulate yeast beta-glucan and bacterial lipopolysaccharide (LPS) showed increased production of lysozyme in the culture supernatants compared to non-treated controls. The increased lysozyme production started at day 3 and was five- to six-fold higher compared to controls at day 6 in culture. Beta-glucan showed an approximate linear dose-response curve between 1 and 250 microg x ml(-1) whereas LPS showed a dose-response curve with a well-defined optimum concentration (10 microg x ml(-1)). The increase in lysozyme activity was accompanied by an accumulation of lysozyme gene transcript in the stimulated cells. Recombinant human tumor necrosis factor alpha, known for its ability to stimulate lysozyme in human macrophages and to elevate respiratory burst activity of rainbow trout macrophages, failed to stimulate lysozyme production of Atlantic salmon macrophages. Macrophages isolated from fish suffering from a non-lethal Ichthyobodo necator infection displayed a highly increased ability to produce lysozyme in response to both beta-glucan and LPS. As in higher vertebrates, lysozyme production may reflect the differentiation stage of the Atlantic salmon macrophages as well as a direct activation of lysozyme gene transcription by biological response modifiers. The rather late increase in lysozyme production induced by beta-glucan and LPS may thus be explained by stimulation of differentiation of the macrophages in culture eventually combined with direct activation of transcription of the lysozyme gene.

It has been found that certain antineoplastic drugs impart their function with a distinct duality. Besides being tumoricidal, they are capable of acting as immunopotentiator. This led us to investigate the effect of cytosine arabinoside (CA), vincristine sulphate (VS), cyclophosphamide (CS), mitomycin C (MMC), hydroxy urea (HU) and lipopolysaccharide (LPS) on a macrophage cell line P388D1. Supernatants collected from P388D1 cells treated with CA, VS, CS, MMC, HU or LPS demonstrated enhanced production of tumor necrosis factor (TNF) confirmed by bioassay on L929 tumor target cells and increased interleukin-1 (IL-1) production by standard thymocyte proliferation bioassay. Also, supernatants showed increased amounts of nitric oxide and lysozyme using Griess reaction and reduction in turbidity of Micrococcus lysodeikticus, respectively. The above findings demonstrate that these drugs may be used not only as chemotherapeutic agents but also as macrophage-activating agents.

The mouse CD5 positive pre-B cell line SPGM-1 can be induced to switch its lineage commitment towards macrophage differentiation by treatment with a combination of phorbol ester and a calcium ionophore. When cultured with these reagents the pre-B cells ceased to proliferate and rapidly became adherent to plastic surfaces. This morphological change was accompanied by the loss of pre-B cell-specific surface markers, such as PB76 and most prominently the mu heavy chain of the immunoglobulin receptor complex. In addition, the mRNA of the surrogate light chain lambda 5 disappeared while the induction of lysozyme mRNA could be detected. Differentiated SPGM-1 cells phagocytosed latex beads and showed nonspecific esterase activity. The high efficiency and speed of differentiation in this cellular system makes SPGM-1 a highly suitable model for studying the phenomenon of lineage switching during hemopoesis.

The effect of acyclovir (ACV) treatment on selected functions of human blood-derived macrophages was examined. ACV was not cytotoxic when applied in a wide range of concentrations. Only minor effects on macrophage functions were observed when cells were treated with therapeutic concentrations of ACV:phagocytosis and the production of interferon and tumor necrosis factor were slightly enhanced, while the production of lysozyme was reduced, in a dose-dependent manner. Interferon production was also reduced in the presence of high concentrations of ACV.

Murine cultured microglial cells were immortalized after infection with a v-raf/v-myc recombinant retrovirus. This immortalized cell line (BV-2) shares properties with body macrophages with respect to the antigen profile, their phagocytic capacity and antimicrobial activity. BV-2 cells are not constitutively able to kill tumor cells in vitro, but acquire antitumor activity following an increase in [Ca++]i. BV-2 cells, like microglial cells, are however, distinct from peripheral macrophages by their expression of inwardly rectifying K+ channels in concert with a lack in outwardly rectifying K+ channels and the formation of spineous processes. The BV-2 cell line thus represents a suitable model for in vitro studies of activated microglial cells.

This study demonstrates the induction of lysozyme mRNA expression in situ in tissue macrophages (M phi) of mice following in vivo stimulation. The resting resident tissue M phi of most tissues do not contain enough lysozyme mRNA to be detected by in situ hybridization using 35S-labeled RNA probes. Following Bacille Calmette Guerin or Plasmodium yoelli infection, however, M phi recruited to liver and spleen hybridize strongly to the lysozyme probe. Within 24 h of infection, cells found in the marginal zone of the spleen begin to produce lysozyme mRNA. This response is also evoked by a noninfectious agent (intravenously injected sheep erythrocytes), and is possibly the result of an early phagocytic interaction. Later in the infection, other cells in the red and white pulp of the spleen, and cells in granulomas in the liver, become lysozyme-positive. Kupffer cells are rarely lysozyme-positive. Lysozyme mRNA levels in liver granulomas remain relatively constant during the infection, and lysozyme is produced by most granuloma cells. This contrasts with tumor necrosis factor alpha (TNF alpha) mRNA, which is produced by fewer cells in the granuloma, and which can be massively induced by lipopolysaccharide administration. The production of lysozyme, previously considered a constitutive function of M phi, is therefore an indicator of M phi activation in vivo, where immunologically specific and nonspecific stimuli both stimulate lysozyme production at high levels in subpopulations of cells occupying discrete anatomical locations.

Fibronectin secreted by macrophages may contribute to the development of pulmonary fibrosis. Prostaglandins are important regulators of macrophage metabolism whose role in the regulation of fibronectin production is not known. In this study, we examined the effects of PGE1 and indomethacin on human monocyte-derived macrophages exposed to these agents in culture for 10 to 14 days. Indomethacin (10 micrograms/ml) reduced the ratio of supernatant fibronectin to adherent cell DNA by 32%, p < 0.01, and reduced lysozyme/DNA by 29%, p < 0.0001. Exogenous PGE1 (1 ng/ml) did not affect fibronectin, but increased lysozyme/DNA by 27%, p < 0.01. In additional experiments, supernatant fibronectin and total protein synthesized in the presence of 3H-leucine were measured. Indomethacin (10 micrograms/ml) had no effect on total supernatant protein radioactivity, but reduced fibronectin/DNA by 33%, p < 0.001, and reduced fibronectin/total protein by 19%, p < 0.01. Since indomethacin increases macrophage secretion of plasminogen activator and interleukin-1, these experiments add to the evidence that specific secretory products of macrophages are regulated independently. We conclude that indomethacin at 10 micrograms/ml decreases the production of fibronectin and lysozyme by monocyte-derived macrophages. The modest size of the effect, and its absence at lower doses of indomethacin, indicate that prostaglandins are unlikely to have a major role in the regulation of macrophage production of fibronectin.

FDP produces an increase of serum lysozyme concentration which may be related to stimulation of the phagocytic activity. Mice macrophages in vitro produce extracellular and intracellular LSZ (lysozyme) and FDP (fructose-1-6-diphosphate) increases this production. Also in vivo FDP stimulates the macrophages intracellular lysozyme production. The toxic activity in vitro and the protection in vivo against Staphylococcus pyogenes after FDP administration can also be related to macrophage stimulation.

The ability of exogenous prostaglandins to inhibit complement production (CP) by monocytes and macrophages (M phi) suggests that endogenous arachidonic acid metabolites produced by these cells may also regulate their rate of CP. We assessed the regulatory influence of endogenous metabolites on CP by M phi utilizing exogenous arachidonic acid and serum-opsonized zymosan as stimulators of production of cyclooxygenase and lipoxygenase metabolites. The results of this study show that (i) the inhibition of CP caused by both agents is is independent of arachidonic acid metabolites, suggesting that endogenously produced metabolites do not influence CP, and (ii) arachidonic acid and serum-opsonized zymosan inhibit production by independent mechanisms.

UICC, other well-defined asbestos samples and different man-made mineral fibers (MMM) such as glass fiber and synthetic amphibole asbestos were studied in vitro by using rat and guinea pig lung macrophages. These samples had relatively narrow length and diameter spectra. Most of the fiber samples were added to the cultures on a gravimetric basis, although some were added on a numerical basis. Electrocorundum and DQ12 (Dorentruper Quartz) were used as controls at comparable gravimetrical concentrations. The assays used were the release of lactate dehydrogenase (to demonstrate plasma membrane permeability) and the release of beta-glucuronidase (to indicate lysosomal permeability). Carbohydrate metabolism was monitored by the measurement of lactic acid production and, as one of the tests for macrophage function, the production of lysozyme was determined. The phagocytic ability of the cells was measured, after the addition of opsonized zymosan, by bioluminescence following luminol enhancement. Only some results could be evaluated, however, due to technical difficulties. A length- and dose-dependent cytotoxicity of the fibers was found in this system which was similar to that previously described with permanent cell lines. No great differences were found between fibers having different physicochemical compositions if their geometric dimensions were similar. Long, very thin fibers of glass, chrysotile, crocidolite and synthetic fluoroamphiboles were all toxic in the test system.

To explore mechanisms of drug-induced alterations in local host defenses in the lung, the capacity of guinea pig alveolar macrophages to synthesize the second (C2) and fourth (C4) components of complement was studied following treatment with cyclophosphamide and cortisone acetate. Administration of either drug significantly inhibited biosynthesis of C2 and C4 within 1 day. After 1 week of treatment, local complement synthesis was inhibited approximately 80%, although serum levels of the corresponding proteins were normal.

Sodium thioglycollate-induced peritoneal macrophages from C3D2F1 mice were examined for their ability to produce and secrete lysozyme in vitro. Lysozyme was quantitated by use of a "lysoplate assay", in which lyophilized Micrococcus lysodeicticus suspended in agar were lysed by the enzyme. The ability to secrete lysozyme decreased in mice bearing a subcutaneous or intraperitoneal mastocytoma P-815 with progressive tumour growth.

The synthesis of C3, C2, factor B (B) C1-inhibitor and lysozyme has been studied in monocytes and macrophages isolated from the synovial fluids of patients with rheumatoid arthritis. Concentrations of all 5 proteins in culture supernatants were measured by the sandwich ELISA technique. Kinetic studies showed that only lysozyme and C3 could be detected in monocyte culture supernatants on the first day of culture, whereas C2, B and C1-inhibitor were not present until the third day. In contrast all 5 proteins could be detected in the supernatants of macrophage cultures on day 1. In both monocyte and macrophage cultures synthesis of lysozyme and C1-inhibitor continued throughout the culture period, whereas synthesis of C2, B and C3 appeared to be reduced after the fifth day in culture. Quantitative studies showed that the secretion rates of lysozyme (4,700 X 10(3) molecules/cell/hr) was similar in monocytes and macrophages. Synthesis rates for all 4 complement components in monocyte cultures were less than 0.2% of that for lysozyme. Although the synthetic rates were higher in macrophages, even then they constituted less than 2% of the rate for lysozyme. Synthetic rates for complement components, but not lysozyme, were increased by BSA-anti-BSA antigen-antibody complexes and reduced by serum-treated complexes. Although the functional activity of monocyte B was similar to that for serum, the activity of monocyte C2 was 5 times that of serum C2. As C42 formed with monocyte C2 had a half-life of 13.5 min at 30 degrees C, compared with 4.5 min for the enzyme formed with serum C2, it is probable that monocyte C2 is oxidized by the oxygen products of these cells.

Varying culture medium oxygen tension from one-half to twice the normal concentration did not alter adult or cord blood monocyte-derived macrophage secretion of lysozyme or total protein. Lowering oxygen tension did result in significant increases in fibronectin concentration in both adult and cord blood macrophage culture supernatants. Dexamethasone (10(-7) M) completely blocked adult macrophage but had no effect on cord blood macrophage fibronectin release in response to lowering oxygen tension. These results indicate that human macrophages selectively regulate fibronectin production in response to changes in oxygen tension in vitro.

Normal myeloid and MGI(+)D(+) clones of myeloid leukemic cells can be induced for Fc and complement component 3 rosettes, lysozme, and mature macrophages and granulocytes by a protein with macrophage- and granulocyte-inducing (MGI) activity, whereas MGI(+)D(-) clones can be induced by this protein for rosettes and lysozme but not mature cells. Lipopolysaccharides (LPS) from different bacteria induced the appearance of rosettes, lysozyme, and macrophages in some MGI(+)D(+) clones but did not induce any of these changes in MGI(+)D(-) clones. Lipid A gave the same results as LPS. Incubation of MGI(+)D(+) cells with LPS also induced an MGI activity detectable in the culture medium. This activity behaved like MGI in inducing (i) rosettes, lysozyme, and mature cells in MGI(+)D(+) leukemic cells including a clone resistant to LPS, (ii) rosettes and lysozyme in MGI(+)D(-) leukemic cells, and (iii) differentiation of normal myeloid cells to mature macrophages and granulocytes. This activity was induced in MGI(+)D(+) cells by LPS before the induction of rosettes or lysozyme. The results indicate that the lipid A portion of LPS indirectly induces differentiation of MGI(+)D(+) myeloid leukemic cells by inducing MGI protein. It is suggested that induction of specific regulatory proteins may be a more general mechanism for the induction of differentiation by surface-acting compounds.

Fourteen continuous tissue culture cell lines derived from mouse, rat, or human granulocyte-macrophage cancers were studied for expression of spontaneous and inducible markers of differentiated cells. Five cell lines (two mouse, two rat, and one human) synthesized myeloperoxidase spontaneously, and a fifth mouse line showed biochemically inducible enzyme. Twelve lines (6 mouse, 3 rat, and 3 human) produced lysozyme (muramidase), and all had detectable beta-glucuronidase. Superoxide generation was detected in one mouse, and three human cell lines following stimulation with phorbol myristate acetate. Maturation to differentiated polymorphonuclear leukocyte or macrophage morphology was induced in 3 cell lines (2 mouse and 1 human) following culture in diffusion chambers in total-body-irradiated rats. In vitro morphological differentiation was inducible in one (mouse) cell line exposed to casein, thioglycolate, or plasma from irradiated rats or mice. These findings indicate that mammalian cell lines derived from granulocyte-macrophage cancers stably express several combinations of differentiation markers. The patterns of expression of these markers did not always correlate with the morphological stage of differentiation.

The chicken lysozyme gene is constitutively active in macrophages and under the control of steroid hormones in the oviduct. To investigate which DNA elements are involved in the control of its expression in macrophages we performed transient DNA transfer experiments with two different types of plasmids: 5'-deletion mutants of the upstream region of the chicken lysozyme gene and different fragments from this area in front of the thymidine kinase promoter (herpes simplex virus), each placed in front of the CAT (chloramphenicol acetyl transferase) coding sequence. Two enhancers (E-2.7 kb and E-0.2 kb) were characterized. They are active in macrophages, but not in chicken fibroblasts. Furthermore a negative element (N-2.4 kb) was identified, which is active in fibroblasts and promyelocytes, but not in mature macrophages. The combined action of all three elements contributes to the observed lysozyme gene activities: no activity in fibroblasts, moderate activity in promyelocytes and high activity in mature macrophages.

Methods for the isolation and culture of macrophages from normal human intestine are described. After disaggregation using sequential treatments of dithiothreitol, ethylenediaminetetraacetate, collagenase, and deoxyribonuclease, Percoll density gradients (1.064 SG) produced single cell suspensions from which macrophages were readily purified by adherence to plastic. Macrophages were characterized by morphology, phagocytosis, cytoplasmic staining for nonspecific esterase, presence of membrane Fc receptors, Ia-like antigens, and lysozyme synthesis and secretion. In 10 separate experiments, recovery of viable mononuclear cells was 2.9 +/- 0.6 x 10(6) cells/g of mucosa. Thirty percent of these cells were phagocytic. After adherence to plastic, the macrophage recovery was 1.05 +/- 0.2 x 10(6) cells/g mucosa and 90% +/- 0.4% of the adherent cells in the monolayer were phagocytic. Fifty-five percent of the adherent cells showed Fc receptors for immunoglobulin G, while 94% expressed the Ia-like antigen on their membrane. The successful isolation and culture of human intestinal macrophages in large numbers will allow detailed study of their role in the mucosal immune response in health and disease.

Human blood-born monocytes have been cultivated on the hydrophobic side of Teflon foils (fluorinated ethylene propylene copolymer) using human pooled AB-serum as essential growth factor. At any stage of culture these in vitro maturing macrophages can easily be detached from the Teflon membrane and subjected to further experimentation. Once established, primary macrophage cultures can be maintained in medium containing 10% FCS for up to 3 months. Lysozyme secretion increased more than 10-fold during the sequential process of monocyte transformation into macrophages and correlates with cell number and stage of maturation. The ability to inhibit growth of human permanent tumor cell lines also developed during macrophage maturation. Studies on the cells of 22 healthy donors revealed a reproducible activity of mature macrophages against K562 and MOLT4 tumor cells. Our system will facilitate investigations on various aspects of human macrophage differentiation and function.

The cultured mouse macrophage-like cell line Mm-1 synthesizes and secretes lysozyme continuously like normal macrophages. Culture of the cells in the presence of prostaglandin D2 for 3 days strongly inhibited their production of lysozyme activity. Prostaglandin D2 caused dose-dependent inhibition of the activity: 1 X 10(-6) M prostaglandin D2 caused about 50% inhibition. Inhibition by prostaglandin D2 was not related to cytotoxicity and was reversible. The rate of synthesis of lysozyme protein was measured by culturing Mm-1 cells with radioactive amino acids and then immunoprecipitating the protein. At the concentrations used, prostaglandin D2 inhibited the synthesis of lysozyme dose-dependently, but did not suppress the synthesis of total protein. Of the various types of prostaglandin, only prostaglandin D2 inhibited the production of lysozyme in Mm-1 cells. Moreover, prostaglandin D2 did not inhibit the production of other lysosomal enzymes, such as acid proteinase, acid phosphatase and beta-glucuronidase, and did not affect Fc receptors on the cell surface, adherence of cells to the culture dish or the cell morphology. These results indicate that prostaglandin D2 specifically inhibits the synthesis of lysozyme in Mm-1 cells. When Mm-1 cells were cultured for 3 days in the presence of the ethyl acetate extract from the culture medium in which Mm-1 cells had been cultured with prostaglandin D2 for 3 days, the production of lysozyme activity of Mm-1 cells was also markedly inhibited by the extract. After the incubation of prostaglandin D2 for 3 days with Mm-1 cells, less than 10% of the initial prostaglandin D2 remained and two major metabolites appeared. These results suggest that the metabolites of prostaglandin D2 were involved in the inhibitory action of prostaglandin D2 in Mm-1 cells.

Cultured thioglycollate-stimulated peritoneal macrophages synthesize, accumulate, and continuously release high levels of plasminogen activators for at least 4 days whereas cultures of unstimulated macrophages do not; the higher specific catalytic activity of released vs. cell-associated enzyme suggests that the plasminogen activators are actively secreted. The major macrophage plasminogen activator is a serine protease of mol wt 48,000, and thus resembles the comparable enzyme released by virally transformed fibroblasts. Macrophages release a second plasminogen activator of mol wt 28,000 that is also a serine enzyme. The secretion products released by stimulated and unstimulated macrophages have been compared by SDS-polyacrylamide gel electrophoresis after chemical labeling with (3)H-DFP or biosynthetic labeling with (14)C-amino acids. These procedures show that some proteins are formed in both cultures, whereas others are uniquely secreted by each type of macrophage. The serine enzymes released by the two kinds of macrophages differ in specificity and electrophoretic mobility.

Tissue macrophages produce several proteins of the complement system. The mechanisms that regulate this process are poorly understood. The established ability of certain prostaglandins to influence macrophage secretory activity suggests that these lipid mediators may also modulate complement production (CP). Using the guinea pig peritoneal macrophages, we determined the effects of selected prostaglandins on in vitro CP and found that PGE2 inhibited production of complement proteins but not lysozyme; the response of elicited and resident peritoneal cells to PGE2 was identical; and PGE1, PGF2 alpha, and PGI2 had no detectable effect. PGE2 may contribute to regulation of CP in vivo.

A system has been developed with clones of mouse myeloid leukemic cells in culture to study the induction of synthesis and secretion of lysozyme in relation to other steps in myeloid cell differentiation. Lysozyme was initially absent in all the clones studied. The different clones can be divided into three types according to their ability to be induced to undergo normal cell differentiation by the protein inducer MGI (macrophage and granulocyte inducer). One type of clone that can be induced by MGI to form Fc and C3 receptors and differentiate to mature macrophages and granulocytes (MGI+D+) was also induced by MGI to synthesize and secrete lysozyme. Lysozyme was induced after Fc and C3 receptors, and labeling with 35S-methionine has shown that the induced lysozyme was newly synthesized. MGI+D+ clones were also induced to synthesize and secrete lysozyme by dexamethasone, prednisolone, cytosine arabinoside, or thymidine and in one of four clones by dimethylsulfoxide but not by sodium butyrate. Inhibition of cell multiplication by itself was not sufficient to induce lysozyme synthesis. A second type of clone which can be induced by MGI to form Fc and C3 receptors but not mature cells (MGI+D-) was more weakly inducible by MGI for lysozyme and was not inducible by any of the other compounds. A third type of clone (MGI-D) MGI for receptors or mature cells. One of four MGI-D- clones was induced to synthesize but not secrete lysozyme by dexamethasone together with sodium butyrate, but there was no lysozyme induction by MGI or any of the other compounds separately. The different clones maintained their different properties for at least 6 months in culture. The results indicate that clones with different hereditary blocks in the ability to be induced to differentiate by specific compounds can be used to dissect the process of myeloid cell differentiation, that the sequence of differentiation is induction of Fc and C3 receptors leads to lysozyme leads to mature cells, and that there are separate controls for these developmental steps.

Mouse myeloid leukemia cells can be induced to differentiate into macrophages in vitro by 1 alpha,25-dihydroxyvitamin D3, the active form of vitamin D3. The minimal concentration of 1 alpha,25-dihydroxyvitamin D3 to induce the cell differentiation was 0.12 nM. The degree of cell differentiation in various markers induced by 12 nM 1 alpha,25-dihydroxyvitamin D3 was nearly equivalent to that induced by 1 microM dexamethasone, the most potent known stimulator. Among several markers of the differentiation by 1 alpha,25-dihydroxyvitamin D3, phagocytic activity was induced within 24 hr, and this was followed by induction of lysozyme and locomotive activities. Similar changes were also induced by 0.01-1 microM 1 alpha-hydroxyvitamin D3. 25-Hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 showed only weak inducing activity. These results suggest the possibility that, in addition to its wellknown biological activities in enhancing intestinal calcium transport and bone mineral mobilization, 1 alpha, 25-dihydroxyvitamin D3 is involved in the differentiation of bone marrow cells.

The synthesis and secretion of plasminogen activator by cultured macrophages can be induced and stimulated by concanavalin A and by phorbol myristate acetate, and inhibited by such agents as glucocorticoids, mitotic inhibitors and compounds affecting cAMP metabolism. By the manipulation of stimulatory and inhibitory influences, enzyme production can be modulated continuously over a 200 fold range. In the same way, the proportion of cells that secrete detectable levels of enzyme can be varied from 1-90%. No comparable modulation of lysozyme or acid hydrolase production is observed under the same conditions. These results suggest that the physiological control of macrophage plasminogen activator production is achieved by the interacting effects of mutually antagonistic stimuli; this emphasizes the utility of this enzyme for the study of regulatory phenomena, including those relating to inflammation.

Pure cultures of three types of mononuclear phagocytes-mouse peritoneal macrophages, unstimulated or after thioglycollate stimulation, and human monocytes-synthesize and secrete large amounts of lysozyme in vitro. The macrophage lysozyme is indistinguishable from authentic lysozyme in its ability to lyse M. lysodeikticus, inhibition by specific antisera, a similar size of 14,000 and cationic charge. Lysozyme secretion in culture is characterized by a large net increase in total lysozyme, 4-20-fold in 3 h, 75-95% of which is in the medium, and its continued extracellular accumulation over at least 2 wk in culture. Lysozyme is the major (14)C-labeled protein secreted into the medium by both unstimulated and thioglycollate-stimulated macrophages and the 0.75-1 microg produced per 1 x 10(6) cells/day represents 0.5-2.5% of the total cell protein. Lysozyme is a cell-specific marker for mononuclear phagocytes and the PMN, which contains preformed enzyme, since it is absent in lymphoid cells and a variety of fibroblast and epithelioid cell lines. Lysozyme production is also a useful measure of mononuclear phagocyte cell number. The rate of lysozyme production and secretion is remarkably constant for all cell types under a variety of culture conditions. Production by the mouse macrophage increases threefold on the 2nd day in culture and then remains linear with time. Production is optimal at a relatively low serum concentration, but can be maintained, in the absence of serum, in lactalbumin hydrolysate or, at a reduced level in basal media. The production and secretion of lysozyme are independent of the production of macrophage acid hydrolases. Net increase and secretion of lysozyme occur under conditions where acid hydrolases like N-acetyl beta-glucosaminidase, beta-glucuronidase, beta-galactosidase, and cathepsin D are neither accumulated nor secreted. Massive phagocytosis of latex particles has no effect on lysozyme production and secretion. Lysozyme production can be rapidly inhibited by treatment with cycloheximide (0.4 microg/ml) whereas inhibition of its production by colchicine (10(-6) M) occurs only after a lag period of more than 8 h, and is probably due to a secondary effect. These results show that mouse macrophages provide a simple in vitro system to measure lysozyme secretion and its control. These studies also indicate the possible importance of mononuclear phagocytes in the secretion of a variety of biologically active products and in the modification of their environment.

This report presents results using a commercially available serum-free medium, HL-1, to study growth and differentiation of the macrophage-like cell line Mm-1. Mm-1 cells grown in RPMI 1640 plus 5% calf serum were compared with the same cells acclimated to and maintained in HL-1. The two medium formulations are compared for their ability to support replication of the line as well as their ability to support the expression of non-specific esterase production, lysozyme secretion, Fc receptor expression, and phagocytosis of latex beads. The serum-supplemented RPMI promoted growth of Mm-1 cells slightly better than HL-1, but the HL-1 medium consistently supported differentiated functions of the cells 8-10% above the level expressed in serum-supplemented RPMI at the same cell density. Based on these findings and the low endotoxin levels of HL-1, we conclude that the medium is appropriate for refined physiological studies of macrophage functions in this cell line.