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Macrophages are important in innate and adaptive immunity. Macrophage participation in inflammation or tissue repair is directed by various extracellular signals and mediated by multiple intracellular pathways. Activation of group VIA phospholipase A 2 (iPLA 2 β) causes accumulation of arachidonic acid, lysophospholipids, and eicosanoids that can promote inflammation and pathologic states. We examined the role of iPLA 2 β in peritoneal macrophage immune function by comparing wild type (WT) and iPLA 2 β -/- mouse macrophages. Compared with WT, iPLA 2 β -/- macrophages exhibited reduced proinflammatory M1 markers when classically activated. In contrast, anti-inflammatory M2 markers were elevated under naïve conditions and induced to higher levels by alternative activation in iPLA 2 β -/- macrophages compared with WT. Induction of eicosanoid (12-lipoxygenase (12-LO) and cyclooxygenase 2 (COX2))- and reactive oxygen species (NADPH oxidase 4 (NOX4))-generating enzymes by classical activation pathways was also blunted in iPLA 2 β -/- macrophages compared with WT. The effects of inhibitors of iPLA 2 β, COX2, or 12-LO to reduce M1 polarization were greater than those to enhance M2 polarization. Certain lipids (lysophosphatidylcholine, lysophosphatidic acid, and prostaglandin E 2 ) recapitulated M1 phenotype in iPLA 2 β -/- macrophages, but none tested promoted M2 phenotype. These findings suggest that (a) lipids generated by iPLA 2 β and subsequently oxidized by cyclooxygenase and 12-LO favor macrophage inflammatory M1 polarization, and (b) the absence of iPLA 2 β promotes macrophage M2 polarization. Reducing macrophage iPLA 2 β activity and thereby attenuating macrophage M1 polarization might cause a shift from an inflammatory to a recovery/repair milieu., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Trypanosoma cruzi infection, which is the etiological agent of Chagas disease, is associated with intense inflammation during the acute and chronic phases. The pathological progression of Chagas disease is influenced by the infiltration and transmigration of inflammatory cells across the endothelium to infected tissues, which are carefully regulated processes involving several molecular mediators, including adhesion molecules and platelet-activating factor (PAF). We have shown that PAF production is dependent upon calcium-independent group VIA phospholipase A2β (iPLA2β) following infection of human coronary artery endothelial cells (HCAECs) with T. cruzi, suggesting that the absence of iPLA2β may decrease the recruitment of inflammatory cells to the heart to manage parasite accumulation. Cardiac endothelial cells isolated from iPLA2β-knockout (iPLA2β-KO) mice infected withT. cruzi demonstrated decreased PAF production compared to that by cells isolated from wild-type (WT) mice but demonstrated increases in adhesion molecule expression similar to those seen in WT mice. Myocardial inflammation in iPLA2β-KO mice infected with T. cruzi was similar in severity to that in WT mice, but the iPLA2β-KO mouse myocardium contained more parasite pseudocysts. Upon activation, macrophages from iPLA2β-KO mice produced significantly less nitric oxide (NO) and caused lessT. cruzi inhibition than macrophages from wild-type mice. Thus, the absence of iPLA2β activity does not influence myocardial inflammation, but iPLA2β is essential forT. cruzi clearance., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Fatty acid metabolism is perturbed in atherosclerotic lesions, but whether it affects lesion formation is unknown. To determine whether fatty acid synthesis affects atherosclerosis, we inactivated fatty-acid synthase (FAS) in macrophages of apoE-deficient mice. Serum lipids, body weight, and glucose metabolism were the same in FAS knock-out in macrophages (FASKOM) and control mice, but blood pressure was lower in FASKOM animals. Atherosclerotic extent was decreased 20-40% in different aortic regions of FASKOM as compared with control mice on Western diets. Foam cell formation was diminished in FASKOM as compared with wild type macrophages due to increased apoAI-specific cholesterol efflux and decreased uptake of oxidized low density lipoprotein. Expression of the anti-atherogenic nuclear receptor liver X receptor alpha (LXRalpha; Nr1h3) and its downstream targets, including Abca1, were increased in FASKOM macrophages, whereas expression of the potentially pro-atherogenic type B scavenger receptor CD36 was decreased. Peroxisome proliferator-activated receptor alpha (PPARalpha) target gene expression was decreased in FASKOM macrophages. PPARalpha agonist treatment of FASKOM and wild type macrophages normalized PPARalpha target gene expression as well as Nr1h3 (LXRalpha). Atherosclerotic lesions were more extensive when apoE null mice were transplanted with LXRalpha-deficient/FAS-deficient bone marrow as compared with LXRalpha-replete/FAS-deficient marrow, consistent with anti-atherogenic effects of LXRalpha in the context of FAS deficiency. These results show that macrophage FAS deficiency decreases atherosclerosis through induction of LXRalpha and suggest that FAS, which is induced by LXRalpha, may generate regulatory lipids that cause feedback inhibition of LXRalpha in macrophages.

Calcium-independent phospholipase A2 (iPLA2) has been suggested to play an important role in the activation of caspase-1 induced by lipopolysaccharides (LPS). Here, we used pharmacological and genetic approaches to study the role of iPLA 2 in the activation of caspase-1. Bromoenol lactone (BEL), an inhibitor that was originally used to support a role for iPLA2 in the secretion of IL-1 beta, prevented caspase-1 activation induced by LPS and ATP as described, and also activation triggered by Salmonella infection and cytosolic flagellin, which rely on the Nlrc4 inflammasome. Analysis of BEL enantiomers showed that the S-BEL form was more effective than R-BEL in inhibiting the inflammasome, suggesting a role for iPLA2 . However, caspase-1 activation and IL-1 beta secretion and their inhibition by BEL were unimpaired in macrophages deficient in iPLA2 beta. BEL was originally identified as an inhibitor of serine proteases. Consistent with the latter, the serine proteases inhibitors TPCK, TLCK and AAF-cmk prevented the activation of the Nlrc4 and Nlrp3 inflammasomes while pan-cathepsin inhibitors were ineffective. These results indicate that iPLA2 beta is not critical for caspase-1 activation as currently proposed. Instead, the results suggest that serine protease(s) targeted by BEL may play a critical role in the activation of the inflammasome triggered by microbial stimuli.

Class A scavenger receptors (SR-A) participate in multiple macrophage functions including adhesion to modified extracellular matrix proteins present in various inflammatory disorders such as atherosclerosis and diabetes. By mediating macrophage adhesion to modified proteins and increasing macrophage retention, SR-A may contribute to the inflammatory process. Eicosanoids produced after phospholipase A(2) (PLA(2))-catalyzed release of arachidonic acid (AA) are important regulators of macrophage function and inflammatory responses. The potential roles of AA release and metabolism in SR-A-mediated macrophage adhesion were determined using macrophages adherent to modified protein. SR-A-dependent macrophage adhesion was abolished by selectively inhibiting calcium-independent PLA(2) (iPLA(2)) activity and absent in macrophages isolated from iPLA(2) beta(-/-) mice. Our results further demonstrate that 12/15-lipoxygenase (12/15-LOX)-derived, but not cyclooxygenase- or cytochrome P450-dependent epoxygenase-derived AA metabolites, are specifically required for SR-A-dependent adhesion. Because of their role in regulating actin polymerization and cell adhesion, Rac and Cdc42 activation were also examined and shown to be increased via an iPLA(2)- and LOX-dependent pathway. Together, our results identify a novel role for iPLA(2)-catalyzed AA release and its metabolism by 12/15-LOX in coupling SR-A-mediated macrophage adhesion to Rac and Cdc42 activation.

Recent evidence supports a regulatory role for the calcium-independent phospholipase A2 (iPLA2) in the antiviral response of inducible nitric-oxide synthase (iNOS) expression by macrophages. Because two mammalian isoforms of iPLA2 (iPLA2beta and iPLA2gamma) have been cloned and characterized, the aim of this study was to identify the specific isoform(s) in macrophages that regulates the expression of iNOS in response to virus infection. Bromoenol lactone (BEL), a suicide substrate inhibitor of iPLA2, inhibits the activity of both isoforms at low micromolar concentrations. However, the R- and S-enantiomers of BEL display approximately 10-fold greater potency for inhibition of the enzymatic activity of iPLA2gamma and iPLA2beta, respectively. In this study, we show that the iPLA2beta-selective (S)-BEL inhibits encephalomyocarditis virus (EMCV)-induced iNOS expression, nitric oxide production, and iPLA2 enzymatic activity in macrophages in a concentration-related manner that closely resembles the inhibitory properties of racemic BEL. cAMP response element-binding protein (CREB) is one downstream target of iPLA2 that is required for the transcriptional activation of iNOS in response to virus infection, and consistent with the effects of BEL enantiomers on iNOS expression, (S)-BEL more effectively inhibits EMCV-induced CREB phosphorylation than (R)-BEL in macrophages. Using macrophages isolated from iPLA2beta-null mice, virus infection fails to stimulate iNOS mRNA accumulation and protein expression, thus providing genetic evidence that iPLA2beta is required for EMCV-induced iNOS expression. These findings provide evidence for a signaling role for iPLA2beta in virus-induced iNOS expression by macrophages.

Lymphokine (LK)-induced aggregation of peritoneal exudate cells (PEC) has been measured using a quantitative technique. Aggregating activity could be removed from LK preparations by absorption of these with PEC and, in addition, the absorbing PEC, on further incubation themselves aggregated. Absorption of aggregating activity to PEC was rapid, being easily measurable at between 2-5 and 10 min although it was difficult to demonstrate at 30 min. Trypsinized PEC were as effective as normal PEC in absorbing aggregating activity. alpha-L-fucose inhibited LK-induced PEC aggregation and this inhibition showed some specificity in being significantly greater than that obtained with D(+)galactose. However, measurement of the subsequent aggregation of PEC which had been pulse exposed to LK, in the presence of alpha-L-fucose, showed that alpha-L-fucose had no effect on this aggregation. It is concluded that the measurement of aggregating activity can be used to study LK binding to PEC, although the relationship of this binding to the inhibition of aggregation obtained with alpha-L-fucose is not clear.

The possible role of cyclic nucleotides in guinea pig macrophage aggregation, induced by human lymphokine (LK) has been investigated. Small increases were found in guanosine 3'5'-cyclic monophosphate (cGMP), but not adenosine 3'5'-cyclic monophosphate (cAMP), levels of lymphokine aggregated macrophages. Addition of exogenous dibutyryl (DB) cAMP, L-isoproterenol, or theophylline did not induce macrophage aggregation. By contrast, both exogenous DBcGMP and carbamyl-choline induced a macrophage aggregation; although DBcGMP was more effective. In addition, both L-isoproterenol and DBcAMP in the presence of theophylline decreased LK-induced macrophage aggregation, whereas D-isoproterenol and DBcGMP had no effect. The results obtained here are discussed in the context of the previously reported effects of cyclic nucleotides on migration inhibitory factor (MIF) activity and the possible role of these agents in the mechanism of action of MIF.

A study has been made of the activity of interleukin 1 (IL-1) and prostaglandins (PGs) in the culture supernatants from unstimulated and lipopolysaccharide (LPS)-stimulated mycobacteria-induced granuloma cells. Both epithelioid cells from bacillus Calmette-Guerin (BCG)-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas, separated on a fluorescence-activated cell sorter using monoclonal antibody specific to guinea pig macrophages, spontaneously secreted low levels of IL-1 (assayed by thymocyte comitogenic and fibroblast mitogenic activities) into culture supernatants. However, culture supernatants from LPS-stimulated epithelioid cells showed significantly higher IL-1 activity than those from unstimulated cells. In contrast, LPS stimulation of M. leprae granuloma macrophages failed to enhance IL-1 production. Nevertheless, IL-1 activity in the culture supernatants from stimulated mycobacterial granuloma cells of both types was much lower than that from LPS-stimulated peritoneal exudate macrophage culture supernatants. There was no detectable amount of prostaglandin E2 (PGE2) in the culture supernatants from both unstimulated and LPS-stimulated BCG- and M. leprae-induced granuloma cells in comparison to much higher levels of PGE2 produced by unstimulated (0.28-6.2 ng/ml) or LPS-stimulated (greater than 15 ng/ml) peritoneal exudate macrophages. However, BCG granuloma cells either secreted prostaglandin F2 alpha (PGF2 alpha) spontaneously or produced comparable levels of PGF2 alpha to those from peritoneal exudate macrophages on stimulation, while M. leprae granuloma macrophages produced much lower levels of PGF2 alpha.

A systematic ultrastructural study of cells of the MPS across the spectrum of leprosy has been carried out. Graded changes in macrophage ultrastructure from the lepromatous to the tuberculoid poles have been shown. Mycobacteria-filled macrophages in lepromatous leprosy are characterised by long cell processes, whereas in borderline tuberculoid leprosy these cells have a rounded appearance and are mainly characterised by numerous intracellular vacuoles. In borderline leprosy, macrophages have an intermediate appearance. Cells of the MPS containing abundant endoplasmic reticulum were only seen in typical "epithelioid cell" tuberculoid granulomas in "BT in reaction" and in the Mitsuda reaction. Epithelioid cell granulomas in other forms of BT leprosy contained activated macrophages.

Granulomas may be immunologically induced or non-immunologically induced. In immunologically induced granulomas cells of the monocyte-macrophage series take on the appearance of epitheloid cells. Ultrastructurally epithelioid cells may have a secretory appearance with much rough endoplasmic reticulum or take on highly degenerate vesicular appearance. Other epithelioid cells look like activated macrophages. Secretory epithelioid cells may be found associated with acute local inflammation as in borderline tuberculoid leprosy in reaction, the lepromin reaction, following injection of BCG vaccine and in experimental zirconium granulomas. In these situations there may also be strong histological and biochemical evidence of increased fibroblast activity and collagen synthesis. It is suggested that these cells are actively secreting a fibroblast-activating factor. Epithelioid cells may lose their Fc receptors, undifferentiated macrophages in lepromatous leprosy can lose their C3 receptors. It is suggested that in a number of situations granuloma formation may be associated with complement activation through the alternative pathway as in the case of mycobacterial granulomas. Toxic granulomas produced by metals may be caused by C3 being split by plasmin after conversion from plasminogen by activation of the Hageman factor.

Cryostat sections of spinal cord of guinea pigs with chronic relapsing experimental allergic encephalomyelitis (CREAE) were stained with monoclonal antibodies recognising a Strain 13-specific Ia epitope, a non-strain-specific Ia antigen and an 'activated' macrophage determinant. It was found that both Ia antigens and the 'activated' macrophage determinant, observed on infiltrating cells within both perivascular and meningeal compartments, appeared to be modulated during the course of CREAE. This correlated with the neurological symptoms of the disease. Blood vessels and 'glial' cells expressed both Ia determinants. 'Glial' cells also expressed the 'activated' macrophage antigen. These antigens were modulated with the course of the disease.

A method for the quantitative measurement of macrophage aggregation by lymphokine preparations has been described previously. This involved the continuous measurement of the light absorbance of stirred suspensions of guinea pig peritoneal exudate cells (PEC). We now show that using a modified method, both aggregation of normal PEC, induced by preformed lymphokine, and direct aggregation of sensitized PEC with specific antigen can be measured. The method is suitable for the assay of large numbers of samples. Aggregation is shown to be immunologically specific for two antigens (bovine gamma-globulin and ovalbumin) and to be partially inhibited by sugars which inhibit migration inhibitory factor activity (rhamnose and fucose).

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.

Epithelioid cells are cells of the mononuclear phagocyte system found in certain granulomas mainly associated with intense immunological activity. These cells show little phagocytic activity. In certain experimental granulomas such as those produced in guinea pigs sensitive to zirconium, and at sites of intense inflammatory reaction in man, they may contain varying amounts of rough endoplasmic reticulum ("secretory" epithelioid cells). In other situations such as tuberculoid leprosy and in some cases of sarcoidosis they may have the appearance of activated macrophages or take on a multivesicular appearance ("vesicular" epithelioid cells). It is suggested that "vesicular epithelioid cells could develop from "secretory" epithelioid cells by a process of degeneration. In studies comparing granulomas induced in lymph nodes draining the site of intradermal injection of mycobacteria, epithelioid cell granulomas were produced with BCG vaccine, whereas, the granulomas induced by Mycobacterium leprae contained undifferentiated macrophages that contained phagocytosed organisms. The BCG granulomas were in addition characterised by fibroblast infiltration, the presence of collagen and resolution by fibrosis. M. leprae granulomas showed little evidence of fibroblastic activity. Biochemical studies confirmed that BCG granulomas formed new collagen in vitro, whereas this did not take place with M. leprae granulomas. It is suggested that epithelioid cells could play an important role in fibrosis possibly by the secretion of a fibroblast activating factor.

Cells of the mononuclear phagocyte system take on different morphological features and appear to have different functions in immunological and nonimmunological granulomas. In immunological granulomas the appearance is of epitheliod cells. Epitheliod cells are of 2 types. In borderline tuberculoid leprosy granulomas, the appearance is of activated macrophages. Similar activation can be induced by lymphokine which increases respiratory enzyme activity. Other types of epithelioid cell are found in experimental zirconium granulomas. These cells contained rough endoplasmic reticulum, indicating that they may play a secretory role. It is suggested that these cells could play a part in stimulating fibrosis. Other substances that produce nonimmunological granulomas, such as aluminum containing compounds, are directly toxic to macrophages in vitro resulting in the rapid release of cytoplasmic enzymes. These compounds also activate complement, causing C3 conversion and anaphylatoxin production. C3 conversion may be through pathways other than the classical and alternative pathways and does not occur in the absence of plasminogen. Mycobacteria can activate complement through the alternative pathway, suggesting a mechanism for granuloma formation in lepromatous leprosy. Loss of C3 membrane receptors from macrophages in lepromatous leprosy could be produced by feeding peritoneal exudate macrophages with mycobacteria in vitro. This was not just due to phagocytosis, as a similar receptor loss was not obtained when the cells were fed latex particles or zymosan. Epithelioid cells in tuberculoid leprosy and sarcoidosis lose Fc membrane receptors but retain C3 receptors. Thus epithelioid cells can be readily distinguished from other cells of the mononuclear phagocyte series.

EA and EAC receptors have been studied on non-elicited and paraffin-induced macrophages, under a variety of culture conditions in vitro, for up to 7 days. A large decrease in the number of macrophages showing EAC receptors was found after treatment of the cells with BCG, but not "inert" particles such as latex and zymosan. This was reversible by 7 days. In the presence of a toxic material, Al(OH)3, both EA and EAC receptors were partially lost. The results obtained have been related to previous results obtained with cryostat sections of human leprosy skin lesions.

A new machine, the Coulter Channelyzer P128, has been used to measure the mean cell volume of suspensions of normal guinea-pig macrophages. Macrophage volume, expressed in cubic microns, has been examined after various times of storage and subsequently after contact with varying concentrations of lymphokine. It is suggested that this machine improves the speed and accuracy of quantitating one in vitro assay of lymphokine activity.

Controversy exists as to whether Langerhans cells in the epidermis and resting microglia in the brain should be included among cells of the mononuclear phagocyte series (MPS). A monoclonal anti-guinea-pig macrophage antibody has been prepared that is specific for a macrophage membrane antigen and does not react with Fc receptors or Ia antigens. This antibody fails to react with Langerhans cells despite reacting with peritoneal exudate macrophages, alveolar macrophages, Kupffer cells and macrophages in infectious granulomas. It does, however, react with resting microglia in the brain. This could suggest that Langerhans cells, despite a similar bone marrow origin, are not typical cells of the MPS, whereas resting microglia share features with this cell system.

The progressive effect of lymphokine contact on macrophages in vitro has been studied using various quantitative cytochemical techniques. Changes in the physiology of the macrophages have been seen rapidly after lymphokine contact. These appear to correlate with the functional effect of inhibition of migration. After more prolonged contact with lymphokine however, the macrophages exhibit different changes to their physiology and reach a state of enhanced cytochemical activity which has been termed 'activation'. By comparing the rapid effect of lymphokine to the changes seen after prolonged contact it is suggested that one can rationalize the apparent paradoxical effects of these soluble mediators which appear initially to 'turn off' the macrophage and subsequently 'activate' the same cell. In doing so an hypothesis is put forward as to how this mediator might work in vivo.

The nature of the suppressive activity in the peritoneal exudate cells (PEC) of guinea-pigs immunized with dinitrophenyl bovine gamma globulin (DNP50-BGG) was investigated. A method was developed to isolate from the peritoneal exudate large numbers of macrophages. Using density gradient centrifugation on Percoll it was possible to obtain a population of cells which contained over 90% macrophages. This macrophage preparation was found to respond to lymphokine but to be incapable of passively transferring delayed hypersensitivity reactions. When these immune macrophages were transferred into antigen immunized animals, which had been pretreated with cyclophosphamide (CY), the skin reactions were suppressed to the same extent as when the total PEC was transferred. PEC from guinea-pigs immunized with ovalbumin in Freund's incomplete adjuvant did not suppress the skin reactions in CY-pretreated DNP50BGG immunized animals. However, in contrast, macrophages from these animals did suppress the skin reactions in the recipient guinea-pigs indicating that the macrophage suppression was not antigen specific.

The damaging effects of some industrially important Aluminium and Zirconium compounds on guinea-pig macrophages and skin fibroblasts have been investigated. Cell death was measured by the release of lactate dehydrogenase activity. The haemolytic properties of these compounds has also been investigated. There is a correlation between the ability of these compounds to form granulomas after intradermal injection in the guinea-pig, their toxic effects on macrophages and fibroblasts and their haemolytic properties. A possible mechanism of toxicity of these compounds towards macrophages and fibroblasts is suggested.

Macrophage aggregation (measured by a light-scattering technique) induced by unpurified lymphokine preparations has been investigated. This has been shown to be Ca2+ or Mg2+ dependent and to be inhibited by a number of agents known to affect cellular physiology and biochemistry. The divalent cation ionophore A23187 causes a macrophage aggregation superficially similar to that induced by lymphokines, although differences were found. It is suggested that a biochemical response by the macrophage is required for aggregation induced by lymphokines, and thus measurements of macrophage aggregation allow a study of the early events in the response of macrophages to lymphokines.

Summary Intracellular mycobacteria release cell wall glycolipids into the endosomal network of infected macrophages. Here, we characterize the glycolipids of Mycobacterium bovis BCG (BCG) that are released into murine bone marrow-derived macrophages (BMMØ). Intracellularly released mycobacterial lipids were harvested from BMMØ that had been infected with 14 C-labelled BCG. Released BCG lipids were resolved by thin-layer chromatography, and they migrated similarly to phosphatidylinositol dimannosides (PIM2 ), mono- and diphosphatidylglycerol, phosphatidylethanolamine, trehalose mono- and dimycolates and the phenolic glycolipid, mycoside B. Culture-derived BCG lipids that co-migrated with the intracellularly released lipids were purified and identified by electrospray ionization mass spectrometry. When delivered on polystyrene microspheres, fluorescently tagged BCG lipids were also released into the BMMØ, in a manner similar to release from viable or heat-killed BCG bacilli. To determine whether the released lipids elicited macrophage responses, BCG lipid-coated microspheres were delivered to interferon gamma-primed macrophages (BMMØ or thioglycollate-elicited peritoneal macrophages), and reactive nitrogen intermediates as well as tumour necrosis factor-alpha and monocyte chemoattractant protein-1 production were induced. When fractionated BCG lipids were delivered on the microspheres, PIM2 species reproduced the macrophage-activating activity of total BCG lipids. These results demonstrate that intracellular mycobacteria release a heterogeneous mix of lipids, some of which elicit the production of proinflammatory cytokines from macrophages that could potentially contribute to the granulomatous response in tuberculous diseases. [ABSTRACT FROM AUTHOR]

A new machine, the Coulter Channelazer P128, has been used to measure the mean cell volume of suspensions of normal guinea-pig macrophages. Macrophage volume, expressed in cubic microns, has been examined after various times of storage and subsequently after contact with varying concentrations of lymphokine. It is suggested that this machine improves the speed and accuracy of quantitating one in vitro assay of lymphokine activity. [ABSTRACT FROM AUTHOR]

Pretreatment of guinea-pigs with cyclophosphamide (Cy) (300 mg/kg) 3 days before cutaneous infection with Leishmania enriettii caused an increased intensity of the lesion at the site of infection and an increase in the incidence of widespread metastases. Decreased levels of circulating antibody were found from the first to fourth week after infection. Decreased delayed type hypersensitivity could only be detected beyond 4 weeks. Peritoneal macrophages obtained from guinea-pigs 3 days after Cy pretreatment showed increased rather than decreased ability to phagocytose L. enriettii, Phagocytosis of L. eariettii by peritoneal macrophages obtained from guinea-pigs pretreated with Cy 24 days previously was normal. It is suggested that more attention should he taken of antibody levels during the early phase of infection and that control of infection could be due to a synergism between antibody arid cell-mediated immunity. Upset in the balance by suppression of either function might lead to the development of widespread metastatic lesions. [ABSTRACT FROM AUTHOR]

The nature of the suppressive activity in the peritoneal exudate cells (PEC) of guinea-pigs immunized with dinitrophenyl bovine gamma globulin (DNP50-BGG) was investigated. A method was developed to isolate from the peritoneal exudate large numbers of macrophages. Using density gradient centrifugation on Percoll it was possible to obtain a population of cells which contained over 90% macrophages. This macrophage preparation was found to respond to lymphokine but to be incapable of passively transferring delayed hypersensitivity reactions. When these immune macrophages were transferred into antigen immunized animals, which had been pretreated with cyclophosphamide (CY), the skin reactions were suppressed to the same extent as when the total PEC was transferred. PEC from guinea-pigs immunized with ovalbumin in Freund's incomplete adjuvant did not suppress the skin reactions in CY-pretreated DNP50BGG immunized animals. However, in contrast, macrophages from these animals did suppress the skin reactions in the recipient guinea-pigs indicating that the macrophage suppression was not antigen specific. [ABSTRACT FROM AUTHOR]