Maximum activities of some key enzymes of metabolism were studied in elicited (inflammatory) macrophages of the mouse and lymph-node lymphocytes of the rat. The activity of hexokinase in the macrophage is very high, as high as that in any other major tissue of the body, and higher than that of phosphorylase or 6-phosphofructokinase, suggesting that glucose is a more important fuel than glycogen and that the pentose phosphate pathway is also important in these cells. The latter suggestion is supported by the high activities of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. However, the rate of glucose utilization by 'resting' macrophages incubated in vitro is less than the 10% of the activity of 6-phosphofructokinase: this suggests that the rate of glycolysis is increased dramatically during phagocytosis or increased secretory activity. The macrophages possess higher activities of citrate synthase and oxoglutarate dehydrogenase than do lymphocytes, suggesting that the tricarboxylic acid cycle may be important in energy generation in these cells. The activity of 3-oxoacid CoA-transferase is higher in the macrophage, but that of 3-hydroxybutyrate dehydrogenase is very much lower than those in the lymphocytes. The activity of carnitine palmitoyltransferase is higher in macrophages, suggesting that fatty acids as well as acetoacetate could provide acetyl-CoA as substrate for the tricarboxylic acid cycle. No detectable rate of acetoacetate or 3-hydroxybutyrate utilization was observed during incubation of resting macrophages, but that of oleate was 1.0 nmol/h per mg of protein or about 2.2% of the activity of palmitoyltransferase. The activity of glutaminase is about 4-fold higher in macrophages than in lymphocytes, which suggests that the rate of glutamine utilization could be very high. The rate of utilization of glutamine by resting incubated macrophages was similar to that reported for rat lymphocytes, but was considerably lower than the activity of glutaminase.

We have recently shown that, following transformation by oncogenic viruses or chemicals, avian and mammalian cells release an enzyme that functions as a plasminogen activator (1-3). The evidence obtained so far indicates that the increased activation of plasminogen determines, at least in part, several of the phenotypic properties of transformed cells; these include colony formation in semisolid media, and the characteristic changes in cell morphology and migration (4). Although the significance of plasminogen activation for cell growth remains to be determined, its association with transformation and neoplasia suggests that the formation of plasminogen activator may also occur during normal cellular proliferation, such as that in the lymphoid and hemopoietic systems.In view of the role of macrophages in lymphocyte activation by lectins and antigens (5, 6), in the production of factors regulating granulocyte differentiation (7), and in chronic inflammation, we have examined pure populations of peritoneal macrophages for production of plasminogen activator. Mononuclear phagocytes are a rich source of acid proteases, such as cathepsins (8), which are thought to play a role in intracellular digestion, but proteases capable of acting extracellularly, at neutral pH, have not been characterized.In this paper, we report that thioglycollate-stimulated mouse macrophages produce and secrete a plasminogen activator in vitro similar to that reported previously from transformed cells. Unstimulated peritoneal macrophages do not produce this enzyme. We have characterized the plasminogen activator secreted into the medium by thioglycollate-stimulated macrophages and have examined the production and release of the enzyme in culture. In addition, we have found that stimulated macrophages m a y also release a variety of proteases that are not formed by unstimulated cells. Materials and MethodsCell Cultures.--Female mice of the NCS (Rockefeller) strain, weighing 25-30 g, were used throughout. Peritoneal macrophages were always harvested without anticoagulants and cul-

The concentrations of ATP and the ATP/AMP concentration ratios were maintained in thioglycollate-elicited mouse peritoneal macrophages incubated in vitro for 90 min in the presence or absence of added substrate: rates of glycolysis, lactate formation and glutamine utilization were approximately linear with time for at least 60 min of incubation. The rate of oxygen consumption by macrophages was only increased above the basal rate (i.e. that in the absence of added substrate) by addition of succinate or pyruvate, or by addition of the uncoupling agent carboxyl cyanide m-chlorophenylhydrazone ('CCCP'); it was decreased by 75% by the addition of KCN. These findings suggest that metabolism of endogenous substrate can provide most, if not all, of the energy requirement of these cells, at least for a short period. The rates of glucose and glutamine utilization by incubated macrophages were approx. 300 and 100 nmol/min per mg of protein respectively. A large proportion of the glutamine that is utilized is converted into glutamate and aspartate, and very little (perhaps less than 10%) is oxidized. Similarly almost all of the glucose that is utilized is converted into lactate and very little is oxidized. This characteristic is similar to that of resting lymphocytes and rapidly dividing cells; in non-proliferating macrophages it may be a mechanism to provide precision in control of the rate of biosynthetic processes that utilize intermediates of these pathways, e.g. purines and pyrimidines for mRNA for the synthesis of secretory proteins and glycerol 3-phosphate for phospholipid synthesis for membrane recycling. No utilization of acetoacetate or 3-hydroxybutyrate by macrophages was detected. In contrast, both butyrate and oleate were oxidized. The rate of [14C]oleate conversion into 14CO2 (1.3 nmol/h per mg of protein) could account for most of the oxygen consumption by incubated macrophages, suggesting that long-chain fatty acids might provide an important fuel in situ. This may be one explanation for the secretion of lipoprotein lipase by these cells, to provide fatty acids for oxidation from the degradation of local triacylglycerol.

Thioglycolate-stimulated mouse peritoneal macrophages secrete a Proteinase which degrades insoluble elastin. There is little elastase activity in cell lysates but the bulk of the enzyme accumulates extracellularly during culture in serum-free medium. The secretion of elastase is sustained for over 12 days in culture and continued secretion of elastase requires protein synthesis. Unstimulated macrophages secrete very little elastase activity but can be triggered to secrete higher levels of this enzyme by phagocytosis and intracellular storage of latex particles. The macrophages elastase is a distinctive proteinase differing from the elastases of pancreas and granulocytes and is distinct from the other secreted proteinases of macrophages, namely, collagenase and plasminogen activator. The macrophages elastase is a serine proteinase and is inhibited by di-isopropyl phosphoro-fluoridate, ovoinhibitor, EDTA, dithiothretiol, and serum. Its activity is little affected by soybean trypsin inhibitor, turkey ovomucoid and chloromethyl ketones derived from tosyl lysine, tosyl phenylalanine, and acetyltetra alanine. Hydrolysis by macrophage elastase of chromogenic ester substrates for pancreatic elastase could not be detected. Elastase secretion by stimulated macrophages exceeds that by primary and established fibroblast cell strains. It is likely that elastase secretion by macrophages plays a major role in the pathogenesis of chronic destructive pulmonary diseases such as emphysema.

In 1922 Alexander Fleming described the remarkable bacteriolytic activity of an enzyme, lysozyme, which was widely distributed in tissues and secretions (1). Lysozyme (muramidase) is a cationic enzyme, tool wt 14,307, which hydrolyses N-acetyl muramic /3-1, 4 N-acetyl glucosamine linkages in the bacterial cell wall (2). Although a great deal is known about its structure and enzymology its function other than in host defence is still poorly understood.High concentrations of lysozyme are found in leukocytes, especially the polymorphonuclear leukocyte (PMN) 1 and rabbit alveolar macrophage (3). Fractionation studies of the rabbit P M N show that 70 % of its intracellular lysozyme is sedimentable and, unlike other hydrolases, it is found in both the azurophil and specific granules of the cell (4). The BCG-induced rabbit alveolar macrophage is able to release a large fraction of its intracellular lysozyme into the medium during phagocytosis (5) and may secrete lysozyme during cultivation in vitro (6). Large amounts of lysozyme accumulate in the serum and urine of patients (7) and animals (8) bearing monocytic leukemia.In this paper we report that mouse peritoneal macrophages and human monocytes synthesize and secrete substantial amounts of lysozyme in culture. We also study factors which influence the rate of lysozyme production and examine the effect of phagocytosis on its secretion. Materials and MethodsCell C u l t u r e s . -Mouse peritoneal macrophages: Female mice of the NCS (Rockefeller) strain, weighing 25-30 g were used. Peritoneal macrophages were harvested, without anticoagulants, by standard procedures (9); the cells were obtained either 4 days after stimulation by intraperitoneai injection of 0.75 ml thioglycollate medium (I0) or from control, unstimulated mice. The cell yield from unstimulated mice was 5-8 X 106 cells, of which 30-40% were macrophages and the remainder lymphocytes; thioglycollate-stimulated mice yielded 15-20 X 106 cells, consisting of 75-90% macrophages and 10-25% lymphocytes.

The injection of thioglycollate medium into the peritoneal cavity of the mouse induces high levels of macrophage fibrinolytic activity due to the production and secretion of a plasminogen activator, a trypsinlike serine protease, which is absent in unstimulated macrophages. Intraperitoneal injection of endotoxin or mineral oil can stimulate only a fraction (<10%) of the fibrinolytic activity of thioglycollate cells, similar to the partial stimulation (<10%) seen 1–2 days after phagocytosis of latex or SRBC by unstimulated macrophages. The endotoxin-stimulated macrophages contain and release relatively low levels of plasminogen activator, but these primed cells can be triggered to produce and secrete high levels of enzyme, by phagocytosis of latex. Under conditions where the plasminogen activator is induced and secreted, there are no effects on the production and/or release of lysozyme or intracellular acid hydrolases, Discovery of a two-stage procedure for inducing macrophage plasminogen activator made it possible to study the role of cell priming and phagocytosis separately. Endotoxin was a more effective priming agent, weight for weight, than lipid A:BSA complex. Secretion of the plasminogen activator was induced only by thioglycollate, or endotoxin and latex. In situ fibrinolysis was induced by these agents and mineral oil, BCG, and fetal calf serum, in decreasing order of effectiveness. Phagocytosis of latex in all cases except thioglycollate stimulation, increased fibrinolytic activity from three- to sixfold. Latex and a variety of other particles such as M. lysodeikticus, aggregated γ-globulin and immune complexes showed dose-dependent stimulation of fibrinolysis by endotoxin-primed macrophages. Although the initial phagocytic trigger was not specific for the substance employed, the ability to induce a sustained response depended on the persistence of the phagocytized particle within the cell. Fibrinolysis and secretion of plasminogen activator continued at high levels for at least 9 days after uptake of latex, a nondigestible particle, whereas plasminogen activator was secreted only transiently after ingestion of rapidly digested M. lysodeikticus. The induction of plasminogen activator secretion provides a mechanism by which the activated macrophage can exert a selective effect on its extracellular environment.

The CD4 antigen is a plasma membrane glycoprotein of^-55 kD that is expressed on most thymocytes and on Th cells in all mammalian species examined, including human, rat, mouse, sheep, and pig (1) . The antigen is also present on rat macrophages (MO) and human monocytes in a similar molecular form (2, 3) . Studies with mAbs (4) have demonstrated that CD4 on Th cells is an important accessory molecule involved in recognition of antigen plus MHC class II molecules during immune responses . In humans, a more recently defined property of the CD4 antigen is to act as the receptor for the AIDS virus (human immunodeficiency virus or HIV) (5) . Although nothing is known concerning the the regulation and functional significance of CD4 expression on MO, its presence is likely to be at least one reason why these cells become infected by HIV (6, 7) . Here we demonstrate that, unlike human and rat M0, mouse Mo do not express the CD4 (L3T4) antigen . This species disparity indicates, firstly, that CD4 may not be essential for Mo function, and secondly, that MO regulate CD4 expression differently from Th cells . Volume 166 August 1987 613-618 Materials and Methods BriefDefinitive ReportAnimals. Specific pathogen-free C57BL/6 mice and AO rats were bred at the Sir William Dunn School of Pathology and both sexes were used between 6 and 12 wk of age .Antibodies. The following rat mAbs to mouse CD4 (L3T4) were obtained as shown and used as tissue culture supernatants at saturation : H129 .19 (IgG2a) (8), RL 172 .4 (IgM) (9), and GK1 .5 (IgG2b) (10), provided by Dr . H . R . MacDonald, Ludwig Institute for Cancer Research, Lausanne, Switzerland; and YTS 191 .1 (IgG2b) and YTA .3 (IgG2b) (11), provided by Dr . S . P . Cobbold, Department of Pathology, Cambridge University, Cambridge, United Kingdom . The noncompeting mouse anti-rat CD4 (W3/25) mAbs, W3/25 (IgGI) and MRC OX-35 (IgG2a) (2), were from the MRC Cellular Immunology Research Unit, University of Oxford, Oxford, United Kingdom . Other mAbs used were MRC OX-21 (IgGI), a mouse mAb to human C3b inactivator (2), and F4/80 (IgG2b), a rat mAb specific for mature mouse Mo (12) .Labeling of Cells with Antibodies . Mouse and rat resident peritoneal MO were purified by adherence for 1 h to bacteriologic petri dishes followed by extensive washing . The adherent cells (>90% Mo by morphology) were detached by a 15-min incubation in calcium-and magnesium-free PBS at 37°C followed by gentle pipetting . Thymocytes

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