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The adjuvant muramyl dipeptide (MDP) has been shown to affect a number of macrophage functions in vitro. We studied the effect of subcutaneous injection of MDP into mice. Cultured peritoneal macrophages from treated mice displayed increased spreading, total cell protein, and specific activity of beta-glucosaminidase a constituent of macrophage lysosomes, and of lactate dehydrogenase. Generation of superoxide anion (O2-) by MDP-treated macrophages stimulated by contact with phorbol myristate acetate was enhanced by over fivefold to levels achieved by macrophages from bacillus Calmette-Guérin-infected mice. The enhancement in stimulated O2- release was noted by 1 h after injection of MDP, peaked by 3 h, and remained high for at least 48 h. Priming for enhancement of O2- release by MDP was similar in athymic nude mice and in normal littermates, suggesting that mature T lymphocytes are not involved in this MDP effect. Priming for enhanced stimulated O2- release, and morphologic and enzymic changes, were not achieved by injection of the D-D stereoisomer of MDP. Phagocytosis of Candida albicans was only slightly greater by macrophages from mice give MDP, but MDP-stimulated cells killed two times more C. albicans in vitro than did cells from untreated animals. When MDP was given 18 h before, simultaneously with, or 24 h after lethal infectious challenge with C. albicans, treated mice were protected compared with controls. These results suggest that injection of MDP effectively and rapidly activates macrophages in the recipient animal. This agent should serve as an important probe of macrophage physiology and, perhaps ultimately, as a means of enhancing host defense in humans.

We explored the relationship between phagocytosis and the triggering of oxidative metabolism using resident, lipopolysaccharide (LPS)-elicited, and bacille Calmette-Guérin (BCG)-activated murine peritoneal macrophages. Sheep erythrocytes (E) coated with IgG [E(IgG)], E coated with IgM and complement [E(IgM)C], and E treated with 1% glutaraldehyde (GE) were used as stimuli. All three types of macrophages released superoxide anion (O2-) during phagocytosis of E(IgG). All macrophage types phagocytosed E(IgM)C and GE but none were stimulated to release O2- during phagocytosis of these particles. Vigorous consumption of oxygen was also stimulated by the ingestion of E(IgG) but not by ingestion of E(IgM)C or GE. E(IgM)C did not scavenge the O2- released from macrophages during phagocytosis of E(IgG) or during exposure to phorbol myristate acetate, and further addition of IgG anti-E antibody to E(IgM)C or GE permitted optimal stimulation of macrophage O2- release by these particles. The capacity of macrophages to ingest E(IgM)C and GE without stimulating the respiratory burst raises the possibility that clearance of particulate matter not opsonized with specific IgG might be achieved without stimulation of the release of toxic oxygen metabolites, and, therefore, without the risk of oxidative damage to the phagocytic cell or surrounding tissue.

After in vitro exposure to lipopolysaccharide (LPS) or muramyl dipeptide (MDP), cultured resident mouse peritoneal macrophages were primed to display enhanced generation of superoxide anion (O2-) in response to stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. Priming with LPS (1 microgram/ml) produced a sevenfold enhancement of PMA-stimulated O2- generation; priming was detected within 30 min and persisted for at least 4 d. Exposure to MDP (1 muM) primed the macrophages to double their O2- release; the response was first observed after 4 h and persisted for at least 3 d. The priming response was not observed with stereoisomers of MDP, which are inactive as adjuvants. LPS and MDP appeared to work directly on the macrophages rather than indirectly by interacting with adherent lymphocytes: (a) Addition of nonadherent cell populations that contained lymphocytes had no effect on the response. (b) The response was normal with cells from nude mice, which lack mature T lymphocytes. (c) Macrophages from C3H/HeJ mice, whose B lymphocytes fail to respond to LPS, were weak in their response to priming LPS; the addition of normal (C3Heb/FeJ) nonadherent cells had no effect on this weak response. (d) The macrophage-like cell line J774.1 also showed enhanced O2--generating capacity after a 4-h exposure to LPS or MDP. The O2--generating capacity of macrophages primed with LPS in vitro was equivalent to that previously observed with cells elicited in vivo by injection of LPS or activated by infection with Bacille Calmette-Guérin. The data suggest that previous exposure to bacterial products could prime macrophages to respond with increased production of toxic oxygen metabolites on contact with invading microorganisms or tumor cells.

Preincubation for 10-30 min with trypsin, pronase, chymotrypsin, or papain primed macrophages to undergo a twofold to sixfold increase in oxidative metabolism, measured as release of superoxide anion or hydrogen peroxide, during stimulation by phorbol myristate acetate or ingestion of Candida parapsilosis. Preincubation of macrophages with inactivated proteases, nonenzyme proteins, or neuraminidase did not affect their oxidase response. Exposure of macrophages to proteases generated at sites of inflammation could prime these cells for a more effective oxidase response to phagocytosis or for greater tissue damage from release of toxic oxygen metabolites.

We studied the capacity of cultured mouse peritoneal macrophages to generate superoxide anion (O(2-)), the initial product of conversion of oxygen to microbicidal species, during phagocytosis of opsonized zymosan or upon contact with the membrane-active agent phorbel myristate acetate (PMA). Macrophages from mice infected with Bacille Calmette-Guerin (BCG) or injected intraperitoneally with thioglycollate broth or endotoxin, released up to 12 times more O(2-) than did resident peritoneal macrophages, depending upon the cell type and whether the stimulus was zymosan or PMA. There was little if any O(2-) release from resting (unstimulated) macrophages. The density of cells on culture dishes was an important variable since crowding of the dish markedly reduced the efficiency of O(2-) production. The enhanced O(2-) release of chemically elicited and infection-activated macrophages was noted after stimulation with a wide range of concentrations of PMA and zymosan, at all time points studied (up to 120 min), and with cells maintained for 140 rain to 16 days in culture. The O(2-) response of resident cells improved twofold to zymosan and ninefold to PMA during the first 3 days in culture. The capacity to release O~ appears to be limited to actively phagocytic cell types: murine macrophage-like tumor lines and cultured human monocytes released O(2-) when stimulated by PMA or zymosan, fibroblast and endothelial lines and embryo-derived cells did not. Activity of superoxide dismutase, which removes O(2-), was not detectable in culture supernates of any cell type, and thus, differences in detectable O(2-) could not be attributed to variations in the release of this enzyme. We conclude that the phagocytosis- associated respiratory burst is significantly enhanced in mononuclear phagocytes obtained ai~r chemical inflammation or BCG infection. Increased capacity to generate O(2-) and other oxygen radicals during phagocytosis could contribute to the improved microbicidal and tumoricidal activity of activated macrophages.

We investigated the capacity of bacterial endotoxin (lipopolysaccharide, LPS) to modify the oxidative metabolic response to membrane stimulation of human neutrophils. Neutrophils were pretreated for 60 min with LPS, 10 ng/ml, then stimulated by exposure to fixed immune complexes, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), or phorbol myristate acetate. Release of superoxide anion (O-2) was up to 7-times greater in cells preincubated with LPS, depending upon the stimulus used. Consumption of oxygen and release of hydrogen peroxide (H2O2) were similarly increased, using FMLP as stimulus. The enhancement was accompanied by a reduction in lag time and an increase in the rate of the response, but the duration of the oxidative events was not changed. The molecular basis for the augmented oxidative response of LPS-pretreated cells was investigated. Preincubation with LPS at 0 degrees C prevented priming, but preincubation in the presence of cycloheximide or chelation of extracellular calcium ion did not. Neutrophils preincubated with LPS had slightly decreased numbers of binding sites and equivalent binding affinity for radiolabeled FMLP. Possible changes in the enzyme responsible for the oxidative burst were analyzed by studying NADPH-dependent generation of O-2 by particulate fractions from cells preincubated with LPS or buffer, then stimulated before cell disruption. The fraction prepared from LPS-pretreated neutrophils exhibited greater release of O-2 over a wide range of concentrations of NADPH. The calculated apparent Km for NADPH was equivalent in the two fractions, but the Vmax was increased 2.5-fold in the subcellular fraction from LPS-pretreated cells. These results suggest that LPS could increase neutrophil-mediated host defense or the tissue damage associated with endotoxemia by enhancing the generation of oxygen metabolites by neutrophils. These results also support the concept that the neutrophil is not an end-stage cell in regard to function or metabolic activity.

We compared phagocytic and metabolic activities of multinucleated giant cells (MGC) and macrophages derived from human monocytes after 9-14 d in culture. Phagocytosis of sheep erythrocytes (E) coated with IgG, of E coated with IgM and complement, and of Candida albicans was comparable in MGC and macrophages. The same percentage of ingested fungi was killed by MGC (24 +/- 4%) and macrophages (21 +/- 5%). Approximately 70% of MGC and macrophages exhibited superoxide-dependent reduction of nitroblue tetrazolium during stimulation. Ia antigen was present on approximately 75% of both cell types. Analysis of cell populations separated by nuclear fluorescence indicated that beta-glucosaminidase, acid phosphatase, and beta-glucuronidase activity per cell was higher in MGC, but specific activity of these enzymes was greater in macrophages. These results suggest that MGC have the capacity to function like macrophages in host defense against infection.

Extent of O-2-release and chemiluminescence, attributed to singlet oxygen, has been compared in human monocytes and neutrophils during phagocytosis, stimulation by the surface-active agent phorbol myristate acetate, or contact with aggregated IgG in a model of immune complex disease. Monocytes generated O-2-and chemiluminescence with each of the three stimuli, although values were significantly less than those of neutrophils from the same individuals. Lymphocytes had no significant activity in either assay with any stimulus. Oxygen metabolites released from mononuclear phagocytes are highly reactive and could play a part in both the beneficial and detrimental aspects of chronic inflammation.

The role of serum factors in the phagocytosis of pneumococci was studied employing a spectrophotometric assay which measures reduced nitro blue tetrazolium (NBT) dye. Dye reduction occurs within the phagocyte shortly after bacterial ingestion as measured by the phagocytic index technique and by the uptake of (125)I-pneumococci. Bacteria prepared with gammaG antibody were not phagocytosed unless a small volume of fresh normal serum was added. Using fresh sera deficient in single complement components, it was demonstrated that the first four components are necessary for optimal bacterial phagocytosis. When highly purified complement components were added to the antibody-coated pneumococci, enhancement of phagocytosis was achieved only with the sequential addition of C1, C4, C2, and C3. Evidence has been presented that human C3 bound to an immune complex exhibits peptidase activity and that this activity is essential for phagocytosis. A heat-labile, dialyzable serum cofactor which enhances C3 peptidase activity enhanced the phagocytosis of pneumococci prepared with purified complement components. A second phagocytosis-promoting cofactor, which is not a complement component, was found to be a heat-labile, 5-6S, beta pseudoglobulin. This protein may stabilize C3 peptidase activity or inhibit enzymatic inactivation of C3.