We separated three macrophage (Mφ) chemotactic factors (MCF-a, -b, and -c) from DNP-ascaris extract-induced and PPD-induced hypersensitivity reaction sites in guinea pigs and highly purified them. MCF-a (mw 150, 000) was generated from serum IgG molecule by neutrophil neutral elastase-like enzyme (mw 27, 000) and -b (mw 14, 000) from C5 molecule by neurophil neutral chymotrypsinlike enzyme (mw 35, 000), and -c (mw 110, 000) was found to exist as a complex of Mφ chemotactic lymphokine (mw 12, 500) and serum α-globulin exuded at the reaction sites. These chemotactic factors were characteristically associated with development of Mφ reactions in the immune tissue injuries. Their Mφ chemotactic activities were confirmed to be trans-species specific. MCF-a and -b were effective for la-negative Mφ of guinea pig and mouse, while MCF-c was active for Ia-positive Mφ of the animals. Thus, further functional analysis of the Mφ was strongly required for fundamental understanding of the immune tissue injuries.

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Contractile activity of myosin-B from porcine leukocytes was regulated by Ca²⁺, which was associated with the level of phosphorylation of its myosin light chain. In another series of experiment, it was found that the myosin-B lost its contractility by washing with 1 mM NaHCO₃. However, the washed myosin-B restored its Ca²⁺-sensitive contractility by the addition of not only a fraction prepared from the NaHCO₃-wash of the myosin-B, but also “native tropomyosin (NT) ” which was the regulatory protein fraction from arterial smooth muscle. From the above results, its was considered that the mechanism of Ca²⁺-sensitive contractility of leukocyte-myosin-B was analogous to that of arterial contractile protein. Some preparations of leukocyte-myosin-B showed the same properties as the washed myosin-B in the Ca²⁺-sensitive contractile activity. In the myosin B, the Mg²⁺-ATPase activity was regulated by Ca²⁺ through “NT” fraction from arterial smooth muscle. On the other hand, leukocyte-myosin and arterial tropomyosin (TM) were purified from leukocyte-myosin B and arterial “NT”, respectively. In a synthetic actomyosin consisting of skeletal actin and leukocyte-myosin, arterial NT enhanced the actinactivated Mg²⁺-ATPase activity of leukocyte-myosin. The enhancement was much higher in the presence of Ca²⁺ and associated with an increase in the level of phosphorylation of myosin light chain. The arterial TM enhanced also the Mg²⁺-ATPase activity of the synthetic actomyosin, but its enhancement was independent of Ca²⁺. The enhancement was lower than that of NT and was not associated with an increase in the phosphorylation of myosin light chain. These results indicate that leukocyte contractile protein is essentially regulated by the level of Ca²⁺-dependent myosin light chain phosphorylation, but its contractility is augumented by TM associated with actin, independent of Ca²⁺

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Crosslinking of monomeric IgG 2 bound to specific receptor on the cell surface of guinea pig macrophages generated the triggering signal for superoxide generation system. Binding experiment of monomeric IgG 2 to macrophages indicated that macrophages had saturable binding sites for monomeric IgG 2. Scatchard analysis of binding experiment showed that macrophage had average of 3×10⁵ binding sites per cell and the association constant of the binding was 8×10⁶M^-1. Binding of monomeric IgG 2 could be detected by the binding of ¹²⁵I-labelled F (ab') ₂ fragment of rabbit antibody specific for guinea pig Fab. Although binding of IgG 2 monomer to Fc_γ receptor did not stimulate superoxide generation, further addition of F (ab') ₂ fragment of anti-guinea pig Fab antibody did induce generation and release of superoxide which was dependent on the dose of cell-bound IgG 2. Addition of Fab monomer fragment of the same antibody did not induce superoxide release. These results show that crosslinking of Fc_γ receptors results in generation of triggering signal for superoxide generation.

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When guinea pig polymorphonuclear leukocytes and macrophages encounter immune complexes, they exhibit phagocytic activity and promote O^-₂ production. The triggering mechanisms of the complexes for inducing these activities were studied with various protease inhibitors. The sucrose density gradient fractionation of homogenized macrophages revealed that diisopropyl fluorophosphate (DFP) blocked the ingestion of soluble antigen-antibody complex, probably the formation of phagosomes since it caused a marked accumulation of the complex in a region between the plasma membrane and lysosomes. On the contrary, p-tosyl-L-lysine chloromethyl ketone accumulated the complex in the lysosomes by inhibiting the intralysosomal digestion of the complex. The enhancement of O^-₂ production from C3 b-zymosan-stimulated polymorphonuclear leukocytes also was inhibited by DFP. The assay of the isolated membrane fractions of the cells for NADPH oxidase demonstrated that DFP inhibited directly the activation process of the enzyme with C3 bzymosan. These results provide strong support to the involvement of a certain serine protease in the triggering mechanisms of immune complexes leading to phagocytosis as well as activation of NADPH oxidase.

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5'-Nucleotidase, alkaline phosphodiesterase, Mg²⁺-ATPase and leucine aminopeptidase are generally reported to be located on the plasma membrane of various mammalian cell types. Among these enzymes, the presence of leucine aminopeptidase (LAP) in neutrophils has not been clear. Therefore, we started with examining the existence of LAP in neutrophils and its subcellular localization with reference to 5'-nucleotidase and alkaline phosphodiesterase. 5'-Nucleotidase was hardly detected in human. Alkaline phosphodiesterase was found in guinea-pig, human and rabbit, but no evidence for the ecto-enzyme was obtained in guinea pig. On the other hand, LAP was observed with all the neutrophils examined and proved to be located as an ecto-enzyme by applying the criteria DePierre and Karnovsky have used to define ecto-enzymes: comparison of the enzyme activity of intact cells with the total cell activity using sonicates, localization of the products of intact cell enzymatic activity and inhibition of intact cell enzyme by a “nonpenetrating” reagent without the inhibition of a soluble cytoplasmic enzyme. Next, the possible functions of LAP were examined. Tuftsin, a phagocytosis-stimulating tetrapeptide, is liberated from leukokinin, neutrophilic γ-globulin with phagocytosis-stimulating activity, by leukokininase on the plasma membrane of neutrophils and these stimulatory effects are not maintained and fall off in time to control levels. On the other hand, the biological activity of tuftsin is reported to be susceptible to commercial leucine aminopeptidase. Therefore, we examined whether LAP in neutrophils acts as a tuftsin-inactivating enzyme or not. Experimental data showed that a tuftsin-inactivating activity was localized as an ecto-enzyme on the plasma membrane and its activity was parallel with the change in the LAP activity which was competitively inhibited by tuftsin, suggesting the possible responsibility of LAP for the tuftsin-inactivating activity of neutrophils.
Phagocytosis is characterized by internalization of large portion of surface membrane of neutrophils and so ecto-enzymes might be interiorized along with plasma membrane and inactivated during phagocytosis. Then, we studied changes in LAP activity during phagocytosis of phagocytic particles. Inactivation of LAP was observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of LAP activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor (s) which are probably activated by the attachement of an opsonized zymosan particle to a specific membrane receptor, probably the C 3 b receptor. Abovementioned results suggested that LAP may modulate the regulation of phagocytic function of neutrophils, which led us to investigate the distribution of the LAP activity among leukocytes other than neutrophils. So-called “professional” phagocytes such as neutrophils, monocytes and macrophages showed higher enzyme activity than lymphocytes and eosinophils in all species examined although the difference was observed in the LAP activity between species. A chemical modification of intact guinea-pig leukocytes with diazotized sulfanilic acid, a poorly penetrating reagent, suggested that LAP was located as an ecto-enzyme in all cell types.

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Muramyl dipeptide (MDP) and lipopolysaccharide (LPS) from E. coli were tested for their ability to influence superoxide anion (O^-₂) release from phagocytes of guinea pigs. Both MDP and LPS alone did not, by themselves stimulate macrophages and polymorphonuclear leukocytes (PMN) to release O^-₂. However, the preincubation of macrophages with MDP or LPS primed the macrophages to release the enhanced amount of O^-₂ when stimulated by cytochalasin E (Cyt E) and wheat germ agglutinin (WGA) . When PMN 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 the enhanced O^-₂ release by MDP.

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Human polymorphonuclear leukocytes release superoxide not only during phagocytosis but also on contact with various surface active agents including chemotactic peptides, calcium ionophore A23187 and phorbol myristate acetate. Superoxide production induced by these soluble stimuli is modulated by potent inactivators of serine proteases, cyclic AMP and cytoskeleton-affecting agents, although the effects of these pharmacologic agents are different according to the stimuli used, suggesting that chemotactic peptides, ionophore A23187 and phorbol myristate acetate may activate the oxidative metabolism of human polymorphonuclear leukocytes through different mechanisms. It is the purpose of this article to review the recent advances in the regulatory mechanisms of superoxide production in human polymorphonuclear leukocytes.

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Superoxide release from polymorphonuclear leukocytes (PMNL) stimulated by concanavalin A was enhanced by cytochalasin (Cyt) A at lower concentrations. However, Cyt A did not stimulate PMNL to release superoxide by itself. These concentrations of Cyt A did not affect the phagocytosis of diisodecyl phthalate emulsion and bacteria. The enhancing effect of superoxide release was reversible, dependent on extracellular Ca, and accompanied by the release of membrane-bound Ca.
Higher concentrations of Cyt A inhibited both superoxide release and phagocytosis irreversibly. These effects were decreased by the preincubation of PMNL with sulfhydryl (SH) agents before the addition of Cyt A and seemed to resemble those of penetrating SH-blocking agents. Cyt A did not affect the activity but the activation of NADPH oxidase. It is suggested that Cyt A acts not only as a SH-blocking agents, but has actions resembling the other cytochalasins.

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Producion of superoxide anion, probably by the action of NADPH oxidase in the cell membrane, was markedly stimulated in guinea pig polymorphonuclear leukocytes by the treatment with membrane-perturbing agents such as cytochalasin D, phorbol myristate acetate, concanavalin A, etc. Anti-microtubular agents, vinblastine and colchicine, further increased the cytochlasin D-or phorbol myristate acetate-stimulated superoxide anion production at a certain concentration range. From the results of experiments to change the intracellular situation, it was assumed that the effect of the anti-microtubular agents was due to the alteration of the state of microtubules around the cell membrane, which might be involved in the regulation of the activity of membrane enzymes. The anti-microtubular agents, however, had little effect on the concanavalin A-stimulated superoxide anion production. The results suggested that the involvement of microtubules in the effect of membrane-perturbing agents was not common but depended on the site of the action of each of the membrane-perturbing agents.

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Both hyperpolarization and depolarization of cell membrane have been observed in neutrophil exposed to various stimuli. With the techniques for the measurement of membrane potential by using the partition of lipophilic probes, we have found that many of stimuli elicit a depolarization in 30 seconds after stimulation. But the change in membrane potential is unlikely to be involved in the initial triggering process leading to activation of several neutrophil metabolisms, because the collaps of membrane potential has no any special implication in cell activation. These membrane potential change may contribute the orientation or pass through of protein or other substance in membrane and may appear to provide the trigger for stimulation of some membrane bound enzymes.

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The oxidase responsible for the“respiratory burst” of phagocytosing polymorphonuclear leukocytes (PMN) was reviewed with recent works on the cellular and subcellular fractions of PMN. (1) In view of the kinetic studies on the oxidase, we concluded that the actual substrate is NADPH but not NADH. (2) NADPH oxidase is located in the plasma membranes. The inhibitory effect of Cibacron-Blue F3GA on the oxidase suggests that the NADPH binding site of the oxidase is located on the inner surface of the plasma membrane. The O^-₂-releasing site seems to be located on the outer surface of the plama membrane. (3) The nitrogen-cavitation method, which pinches off plasma membrane fragments from cells, separated NADPH-oxidase rich and poor vesicles from plasma membranes. The results suggest that activation of PMN either by soluble stimulators or bacteria, leads to a heterogeneous distribution of NADPH oxidase on the plasma membrane. (4) Inhibitors on NADPH oxidase in membrane vesicles are summarized. Some inhibitors suggest that hydrophobic portions of plasma membranes play some role in the O^-₂-generating activity. (5) A fraction of plasma membranes, in which a high specific activity of NADPH oxidase is located, contained not only a cytochrome b but also a FAD-binding protein.

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A phagocytic vesicle fraction with high NADPH-dependent O^-₂-forming activity was obtained in large quantity from pig blood polymorphonuclear leukocytes, phagocytosing oil droplets in the presence of cyanide. The activity of NADPH-dependent 2, 6-dichlorophenolindophenol (DCIP) or ferricyanide reduction was also observed in the vesicles. Increasing the concentration of DCIP, the O^-₂-forming activity decreased and the DCIP-reducing activity enhanced, whereas NADPH oxidation was constant, indicating that electrons from NADPH flow to DCIP on the way to oxygen in the O^-₂-forming system. The O^-₂ formation was completely inhibited by 25μM p-chloromercuriben-zoate, whereas the DCIP reduction was only slightly affected. This suggests that the O^-₂-forming activity may consist of, at least, two components. The O^-₂-forming activity and the DCIP-reducing activity were effectively extracted with a mixture containing deoxycholate and Tween 20 and the specific activiities increased about 10 times by the extraction. Both activities in extract were enhanced 2-hold by the addition of 1μM FAD, suggesting that the flavin is a component of the enzyme system. A mechanism by which the O^-₂-forming system is stimulated was also investigated and results which indicate possible involvement of calmodulin as well as protein-phosphorylating and -dephosphorylating reactions in the mechanism were obtained.

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Leukocytes emit weak light in the visible region after the treatment with various opsonized particular or soluble stimulants. O^-⋅₂production is, kinetically, closely related to luminescence intensity. Luminescence obtained from the opsonaized zymosan-induced system with and wthout added bovine serum albumin has relatively intense peaks at or near 490, 530, 570, and 610-630 nm. Such spectra patterns in the region of 530-630 nm seem similar to those reported for the luminescence of photoproducts formed from indole analogs, assuming a red sift by solvent effect. Luminescence at 490 nm is also involved in an excited indole compound. A possible mechanism of emitter from leukocytes is discussed. The production of ¹O₂ if, it is generated, would be in a minimum amount at physiological pH. Furthermore, because of very short lifetime and high reactivity of ¹O₂ with O^-⋅₂, low physiological significance of ¹O₂ would be considered.

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An oxidative metabolic response is induced in polymorphonuclear leukocytes (PMNs) when the PMN membrane is stimulated by either phagocytizable particles or soluble stimuli. This response can be obtained by measuring the energy released as light (chemiluminescence; CL) . A CL is correlated with metabolic activation of the hexose monophosphate shunt (HMPS), and is also oxygen dependent. This light emission results from generation by PMNs of highly reactive and excited state oxygen metabolites. Cyclic hydrazides, such as luminor (5-amino-2, 3-dihydro-1, 4-phthalazinedione) yield a CL when reacted with a variety of oxidizing agents. In this reaction luminol is oxidized to the electronically excited aminophthalate ion, that relaxes to ground state by photon emission. In attempt to increase the sensitivity of CL assay, luminol was added to the incubating suspension of PMNs. CL was monitored using Aminco Chem-Glo photometer or a liquid scintillation spectrometer (model LS-100C; Beckman Inst.) at ambient temperature. Opsonized zymosan as phagocytizable particles was used and PMA (phorbol myristate acetate), Con A+Cyt E, FMLP, Ca²⁺ ionophor A 23187 as soluble stimuli were used. Luminol-amplified CL assay may be a simple, rapid, reproducible and useful method for diagnosis of chronic granulomatous disease (CGD) and a valuable parameter in assessing the oxidative metabolic and phagocytic capabilities of individuals.

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