Muramyl dipeptide (MDP) and lipopolysaccharide (LPS) from Escherichia coli were tested for the ability to influence superoxide anion (O2-) release from guinea pig phagocytes. Both MDP and LPS alone did not, by themselves, stimulate O2 release by macrophages and polymorphonuclear leukocytes. However, the preincubation of macrophages with MDP or LPS primed the macrophages to release an enhanced amount of O2 when stimulated by cytochalasin E and wheat germ agglutinin. When polymorphonuclear leukocytes were treated in the same way, only LPS showed an enhancing effect. MDP enhanced NADPH oxidase activity of macrophages, which is probably the reason for enhanced O2 release by MDP.Peptidoglycans are structures common to bacterial cell walls. Muramyl dipeptide (MDP), Nacetylmuramyl-L-alanyl-D-isoglutamine, is a minimal structure in peptidoglycans responsible for many activities of bacteria, including immunoadjuvant effect (7). Although the injection of a Freund-type water-in-oil emulsion alone generally induces only macrophage accumulation or sometimes foreign body granulomas in the draining lymph nodes, the incorporation of MDP in the emulsion causes remarkable qualitative and quantitative changes, such as the development of extensive epithelioid granulomas indistinguishable from those evoked by tubercle bacilli and a marked infiltration of polymorphonuclear leukocytes (PMN) (8, 28).MDP activates macrophages to become bactericidal (11), and epithelioid cells appear to be bactericidal against tubercle bacilli (3). However, the mechanism causing macrophages to become bactericidal through MDP is not yet known. It is also not known why PMN accumulate in the MDP-induced granulomas and whether PMN are affected by MDP. To understand these unsolved questions, we investigated, in the present study, the effect of MDP on superoxide anion (O2-) release from macrophages or PMN, because O2 has been known to contribute to microbicidal activity of phagocytes (5,6,17) and to cause the generation of chemotactic factor(s) for PMN (15,25,31). Guinea pigs were used because this animal species has been mostly used for the study of granuloma formation by MDP. Since bacterial lipopolysaccharide (LPS) shares many biological activities with MDP, LPS was also studied in parallel with MDP. MATERIALS AND METHODSReagents. Cytochalasin E (CyE), superoxide dismutase (type I, from bovine blood), ferricytochrome c (type VI, from horse heart), and NADPH were purchased from Sigma Chemical Co., St. Louis, Mo. Wheat germ agglutinin (WGA) was purchased from P-L Biochemicals, Inc., Milwaukee, Wis. Renex 30, polyoxyethylene tridecyl ether, a product of ICI Americas, Inc., Wilmington, Del., was the generous gift of M. Nakamura, Kyushu University; Hanks balanced salt solution (HBSS) was purchased from Nissui Pharmaceutical Co., Ltd., Tokyo, Japan. Medium 199 and fetal bovine serum were purchased from GIBCO Laboratories, Grand Island, N.Y. Lipopolysaccharide (LPS) from Escherichia coli (serotype 0.127:B8, type I) was purchased from Sigma Chemical Co. MDP and its ...

Superoxide generation by guinea pig macrophages and polymorphonuclear leukocytes induced by wheat germ agglutinin, immune complexes or forrnyl-methionyl-leucyl-phenylalanine was inhibited considerably by 3'-deazaadenosine. The pre-exposure of the 3'-dezazaadenosine-treated cells to a small amount of phorbol myristate acetate abolished the inhibitory effect of 3'-deazaadenosine on the generation of superoxide.

Using seventeen human tumor cell lines derived from a variety of tissues, specific binding sites for epidermal growth factor (EGF), a mouse submandibular glandderived growth factor, has been characterized.A significant amount of membranebound EGF receptors, although considerably varied, was demonstrated in all the tumor cell lines studied. Epidermoid carcinoma appeared to have more EGF receptors than adenocarcinoma.One small cell carcinoma of the lung, one choriocarcinoma of the stomach and three bone tumors also possessed EGF recptors comparable to those of epidermoid carcinoma, while one adenoacanthoma of the stomach had less EGF receptors comparable to adenocarcinoma.Among a variety of phorbol esters tested, tetradecanoyl phorbol acetate, a potent tumor promotor, was shown to be the most effective compound in inhibiting 125I-labeled EGF binding to its receptors.Our results indicate that human tumor cells contain varying amounts of membranebound receptors for EGF and that phorbol esters interact with these EGF receptor sites. However, the relationship between EGF receptor sites on tumor cells and cellular proliferation and/or differentiation awaits further study.

KMOE -2/05 cells, derived from a patient with acute erythremia, became benzidine-positive after the addition of cytosine arabinoside (CA). Radioimmunoassays using antihuman hemoglobin antibodies revealed an elevated amount of hemoglobin in the CA-exposed cells over that in the control cells (without CA). Isoelectric focusing of the CA-exposed cell lysate formed benzidine-positive foci in the positions of human adult Hb (HbA) and human fetal Hb (HbF). To determine the types of globin synthesized in the CA-exposed cells, globin chains internally labeled with [3H] leucine were purified by carboxy-methyl (CM)-Sephadex column chromatography, immunoadsorption by Sepharose-coupled antihuman Hb antibodies and Sephadex G-100. The labeled globin chains were finally separated by CM-cellulose chromatography in urea. Two distinct peaks of radioactivity were shown in exactly the same fractions as carrier human globin alpha- and beta-chains. These observations indicate that these KMOE -2/05 cells synthesize HbA.

The effect of phorbol myristate acetate (PMA) on the antibody-dependent cellular cytotoxicity (ADCC) of human polymorphonuclear leukocytes (PMNs) against human erythroleukemic K562 cells was studied by the use of a 3-h 51Cr-release assay. Pretreatment of PMNs with PMA (10 ng/ml) for 60 min resulted in inhibition of subsequent ADCC. This inhibition was dependent on doses of PMA. The effect of pretreatment of PMNs with PMA on O2- generation of the cells was also studied. The ability of the cells to generate O2- was not suppressed, and the expression of Fc receptors on the cell membrane was well preserved. In contrast, the addition of PMA to the ongoing ADCC (5 to 30 min after the start of the ADCC assay) enhanced the activity of the cells for ADCC. This augmentation was abolished by catalase, whereas ADCC itself was not affected by the agent. These results imply divalent effects of PMA on the ADCC of PMNs. The suppression of ADCC activity of PMNs by pretreatment with PMA is not due to inhibition of the reactive oxygen burst of the cells. The augmentation of ongoing ADCC by the addition of PMA is due to secretion of hydrogen peroxide from the cells induced by PMA, and this augmentation occurs only when the interaction between effector and target cells exists through Fc receptor.