Localization of macrophage agglutination factor activity to the gelatin-binding domain of fibronectin

We previously proposed that macrophage agglutination factor (MAggF, a T cell-derived guinea pig lymphokine) is a fibronectin (FN). We now show MAggF binding to gelatin and to peritoneal macrophages is mediated by domains similar to corresponding domains of plasma FN. MAggF activity in lymphokine concentrates prepared by two different methods differed nearly 10-fold in m.w. on gel filtration chromatography. Despite this difference, MAggF dose-activity curves of both preparations were parallel, and MAggF in both preparations bound reversibly to gelatin and to monoclonal anti-guinea pig FN immunoadsorbents. MAggF activity in one preparation was inhibited by the addition of soluble monoclonal antibody specific for the gelatin-binding domain of human FN; inhibitory activity of this antibody was blocked by purified guinea pig plasma FN or partially purified MAggF from the other preparation. Measured MAggF activity of both preparations was reduced in a dose-dependent manner by pretreatment of indicator macrophages with monoclonal anti-human monocyte FN receptor antibody or F(ab')2 fragments or with guinea pig plasma FN. Neither anti-FN receptor antibody nor plasma FN interacted directly with MAggF. Indirect immunofluorescence studies confirmed the presence of uncomplexed plasma membrane receptors for FN on indicator macrophages in MAggF-responsive populations that were able to bind added FN. Our identification of MAggF as lymphokine FN provides a basis for future biochemical analysis of delayed hypersensitivity inflammatory reactions.     

Evidence that NADPH is the actual substrate of the oxidase responsible for the "respiratory burst" of phagocytosing polymorphonuclear leukocytes

The relationship between glucose metabolism and the "respiratory burst" of phagocytosing polymorphonuclear leukocytes (PMN) was studied in a Renex 30-treated cell system of guinea pig PMN by a polarometric technique. Phagocytosing PMN were treated with a detergent (Renex 30) and recovery of respiratory activity was examined by addition of various concentrations of NADP and glucose-6-phosphate (G6P) to determine the availability of endogenously formed NADPH via the hexose monophosphate (HMP) pathway. The oxygen uptake by phagocytosing PMN ceased after the treatment with Renex 30 and was restored by the addition of NADP and G6P. Furthermore, the restoration of oxygen uptake was linearly proportional to the rate of NADPH formation on increase in either NADP or G6P concentration. Resting PMN showed no respiratory activity even in the presence of excess NADP and G6P, in which NADPH was formed at the same rate as in phagocytosing PMN. In a parallel experiment, recovery of respiratory activity was examined in the same system by addition of NAD and glyceraldehyde-3-phosphate (G3P) in that order to clarify whether the respiratory enzyme can utilize NADH formed via the glycolytic pathway. In contrast to the results in the NADPH-forming system, the addition of NAD and G3P induced slight oxygen uptake of Renex 30-treated PMN, but there was no difference in the oxygen uptake between resting and phagocytosis-activated PMN. The results indicated that the primary oxidase responsible for the "respiratory burst" is NADPH oxidase, and that its activity is coupled with glucose oxidation via the HMP pathway without the participation of other metabolic pathways such as glycolysis.     

Macrophage metabolism: activation of NADPH oxidation by phagocytosis

Rabbit and guinea pig peritoneal and alveolar macrophages and rabbit polymorphonuclear leucocytes (PMN) have been tested for their capacity to oxidize NADPH and NADH. In all these cells granule-bound NADPH oxidase is much more active than NADH oxidase, thus confirming our previous observations on human blood and guinea pig PMN. If the phagocytes are challenged with bacteria, the activity of NADPH oxidase is considerably stimulated. The enhancement of the oxidase activity is due to an increase of its V_max and, in the case of the PMN, also to a decrease of the K_m. We conclude that NADPH oxidase might play a relevant role in the metabolic stimulation of both PMN and macrophages by phagocytosis.     

Neisseria gonorrhoeae suppresses the oxidative burst of human polymorphonuclear leukocytes

Symptomatic infection with Neisseria gonorrhoeae (Gc) results in a potent polymorphonuclear leukocyte (PMN)-driven inflammatory response, but the mechanisms by which Gc withstands PMN attack are poorly defined. Here we report that Gc can suppress the PMN oxidative burst, a central component of the PMN antimicrobial arsenal. Primary human PMNs remained viable after exposure to liquid-grown, exponential-phase, opacity-associated protein (Opa)-negative Gc of strains FA1090 and MS11 but did not generate reactive oxygen species (ROS), even after bacterial opsonization. Liquid-grown FA1090 Gc expressing OpaB, an Opa protein previously correlated with PMN ROS production, elicited a minor PMN oxidative burst. PMN ROS production in response to Opa(-) and OpaB+ Gc was markedly enhanced if bacteria were agar-grown or if liquid-grown bacteria were heat-killed. Liquid-grown Opa(-) Gc inhibited the PMN oxidative burst elicited by isogenic dead bacteria, formylated peptides or Staphylococcus aureus but did not inhibit PMN ROS production by OpaB+ Gc or phorbol esters. Suppression of the oxidative burst required Gc-PMN contact and bacterial protein synthesis but not phagocytosis. These results suggest that viable Gc directly inhibits PMN signalling pathways required for induction of the oxidative burst, which may contribute to gonococcal pathogenesis during inflammatory stages of gonorrhoeal disease.     

Beta-endorphin and related peptides suppress phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocytes

In the present study, the immunomodulatory effect of beta-endorphin (beta-E) and shorter pro-opiomelanocortin (POMC) fragments was evaluated by assessing their influence on respiratory burst in human polymorphonuclear leukocytes (PMN). The effect of the peptides (10(-17)M - 10(-10)M) on phorbol myristate acetate (PMA)-stimulated production of reactive oxygen metabolites was measured in a lucigenin-enhanced chemiluminescence (CL) assay. Both POMC peptides with opiate-like activity (i.e. alpha-endorphin (alpha-E), beta-E and gamma-endorphin (gamma-E] and their non-opioid derivatives (i.e. des-TYR1-beta-endorphin (dT beta E), des-TYR1-gamma-endorphin (dT gamma E), and des-ENK-gamma-endorphin (dE gamma E] were tested. With the exception of alpha-E, PMA-stimulated respiratory burst was suppressed by all POMC fragments tested. A U-shaped dose-response relation was observed. Doses lower than 10(-17)M and higher than 10(-8)M were without effect. beta-E and dT beta E both suppressed PMA-induced oxidative burst in human PMN at physiological concentrations (10(-16)M - 10(-10)M). gamma-E and dT gamma E proved to be less potent inhibitors, reaching maximal effect at higher concentrations (10(-12)M - 10(-10)M). DE gamma E exerted an even less pronounced but still significant suppressive effect at the concentration of 10(-10)M. None of the endorphins tested was shown to affect resting oxidative metabolism in the PMN. The modulatory effects of the opioid peptides could not be blocked by the opioid antagonist naloxone (10(-8)M). These data show that fragments derived from the POMC-precursor molecule modulate the activation of PMN by suppressing PMA-stimulated oxidative metabolism and that this activity does not involve a classical opiate-like receptor.     

Sex steroid hormones do not influence the oxidative burst activity of polymorphonuclear leukocytes from ovariectomized cows in vitro

During the periparturient period, dairy cows are subjected to physiological changes that may induce immunosuppression and an increased susceptibility of the animal to bacterial infections such as mastitis. The incidence of clinical environmental mastitis is high during the last period of gestation, at parturition and during the first month of lactation, suggesting a potential influence of sex steroid hormones. Efficient functioning of polymorphonuclear leukocytes (PMN) is necessary during the early phase of infection to clear the mammary gland from invading pathogens. The purpose of this study was to investigate the effect of sex steroid hormones on the oxidative burst activity of isolated PMN from ovariectomized cows. Ovariectomy was performed to minimize the interference of endogenous estrogen and progesterone levels, which are known to vary extensively during the estrus cycle. Isolated PMN were incubated with different concentrations of 17beta-estradiol, estrone or progesterone. A flow cytometric technique was used to quantify the oxidation of intracellular 2',7'-dichlorofluorescin by the oxidative burst system of PMN following stimulation with phorbol myristate acetate. Staurosporine was used as a positive control for our in vitro model. No statistically significant changes in PMN oxidative burst activity were observed at physiological or pharmacological levels of the three sex steroid hormones. A large variation existed in the oxidative burst activity among cows. In an additional experiment, the expression of estrogen receptor alpha and of progesterone receptor in PMN was evaluated immunohistochemically. No specific staining was detected for both receptors in isolated PMN following incubation with different concentrations of sex steroid hormones.     

Cannabinoid receptor-independent suppression of the superoxide generation of human neutrophils (PMN) by CP55 940, but not by anandamide

Cannabinoids have been shown to affect various immune functions. To date, almost no data exist on PMN, which provide the first line antimicrobial defense. The objective of the present study was to investigate the effects of the synthetic dibenzopyrane ligand CP55 940, the endogenous cannabinoid anandamide and methanandamide on the "respiratory burst" of isolated human PMN in vitro. After preincubation with high micromolar concentrations of CP55 940, fMLP-stimulated PMN showed a reduction in superoxide production, whereas the spontaneous burst activity of resting PMN remained unaffected. This inhibitory effect of CP55 940 was not CB-receptor-mediated. In contrast, anandamide and methanandamide did not alter the oxidative microbicidal PMN function.     

Stimulation of neutrophil functions by C5a(desArg): an in vitro model of haemodialysis

Cuprophane membranes during haemodialysis significantly increase the plasma levels of C5a(desArg) (maximal 55 mug C5a(adesArg)/1 blood after 30 min) whereas Hemophane or Polysulphonemembranes induce only low plasma levels of C5a(desArg). C5a(desArg) generated in vitro by yeast incubation of autologous plasma stimulates PMN chemotaxis and oxidative metabolism but has no effect on enzyme release. Preincubation of whole blood with C5a(desArg) causes aggregation and changed oxidative burst activity of the isolated PMN. These changes are similar to those found in cells from patients after haemodialysis with cuprophane membranes. So the elevated plasma levels of C5a(desArg) after haemodialysis explain some of the changes in PMN functions, but additional mechanisms have to be assumed.     

Studies on the effect of soluble lymphocyte products (lymphokines) on macrophage physiology. I. Early changes in enzyme activity and permeability.

Various cytochemical techniques have been used to quantitate the rapid effect of a partially purified, soluble product from lymphocytes (lymphokine) on normal guinea pig macrophages in vitro. Early changes in the utilisation of hydrogen liberated from the hexose monophosphate shunt and on cellular permeability were observed. The ability of the lymphokine to alter hydrogen utilisation was also seen in experiments on cryostat sections of guinea pig liver, suggesting that the cytochemical effects were not predetermined by changes at the membrane level. It is suggested that lymphokine-induced changes within the cell may reduce some biosynthetic activity affecting the cell membrane and this may in part reflect the decreased migrating ability of the cells. Increases in NADPH oxidation after lymphokine contact are discussed in relation to the bactericidal capacity of the cells.     

Characterization of eosinophil cell activation by peptides. Differential effects of substance P, melittin, and FMET-Leu-Phe.

The ability of three different peptides, substance P (SP), FMLP and melittin, to activate eosinophils purified from the peritoneal cavity of human serum-treated guinea pigs was investigated. Degranulation (eosinophil peroxidase, EPO), oxidative burst (O2-), [Ca2+]i mobilization, and arachidonic acid metabolism (thromboxane B2, TXB2) were used as indices of eosinophil activation. SP (100 nM to 100 microM), FMLP (1 to 100 microM) and melittin (10 nM to 100 microM) induced EPO release but only FMLP (1 to 100 microM) led to an elevation of [Ca2+]i. The melittin- and SP-induced EPO secretion occurred at both cytotoxic and noncytotoxic concentrations as assessed by lactate dehydrogenase release. In addition, the effect of SP was not inhibited by the SP analogue (D-Pro4, D-Trp7,9,10)SP(4-11) and SP failed to promote the generation and subsequent release of TXA2. In contrast, FMLP (10 to 100 microM) stimulated the release of TXB2 from guinea pig eosinophils that was selectively inhibited by pretreatment of the cells with BOC-FMLP. On an equimolar basis (1 microM), melittin was approximately fivefold more active at promoting TXB2 release than FMLP. The results indicate that eosinophils respond to the three peptides, SP, melittin, and FMLP in differential fashion. We conclude that activation of guinea pig eosinophils by FMLP is likely to be receptor-mediated whereas the actions of SP and melittin may act through nonspecific peptide-membrane phospholipid interactions.     

Guinea Pig Striatum as a Model of Human Dopamine Deamination: The Role of Monoamine Oxidase Isozyme Ratio, Localization, and Affinity for Substrate in Synaptic Dopamine Metabolism

The kinetic properties of type A and type B monoamine oxidase (MAO) were examined in guinea pig striatum, rat striatum, and autopsied human caudate nucleus using 3,4-dihydroxyphenylethylamine (dopamine, DA) as the substrate. MAO isozyme ratio in guinea pig striatum (28% type A/72% type B) was similar to that in human caudate nucleus (25% type A/75% type B) but different from that in rat striatum (76% type A/24% type B). Additional similarities between guinea pig striatum and human caudate nucleus were demonstrated for the affinity constants (Km) of each MAO) isozyme toward DA. Endogenous concentrations of DA, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were also measured in guinea pig and rat striatum following selective type A (clorgyline-treated) and type B (deprenyl-treated) MAO inhibition. In guinea pig, DA metabolism was equally but only partially affected by clorgyline or deprenyl alone. Combined treatment with clorgyline and deprenyl was required for maximal alterations in DA metabolism. By contrast, DA metabolism in rat striatum was extensively altered by clorgyline but unaffected by deprenyl alone. Finally, the deamination of DA in synaptosomes from guinea pig striatum was examined following selective MAO isozyme inhibition. Neither clorgyline nor deprenyl alone reduced synaptosomal DA deamination. However, clorgyline and deprenyl together reduced DA deamination by 94%. These results suggest that the isozyme localization and/or isozyme affinity for DA, rather than the absolute isozyme content, determines the relative importance of type A and type B MAO in synaptic DA deamination. Moreover, based on the enzyme kinetic properties of each MAO isozyme, guinea pig striatum may serve as a suitable model of human DA deamination.     

Role of lymphokines in regulation of macrophage differentiation

The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages.     

Stimulation of oxidative metabolism of granulocytes by recombinant granulocyte-macrophage-colony-stimulating-factor and a conditioned medium of a urinary bladder carcinoma cell line

Neutrophils (PMN) are the major host defence cells protecting the body against invasion by microorganisms. Products of oxidative metabolism mediate PMN microbicidal and tumoricidal activity, but the mechanisms by which these pathways become activated are not well understood. The colony stimulating factors (CSF) are known to stimulate proliferation and differentiation of committed bone marrow stem cells. These regulators may probably play an important role in non specific resistance to infections. We studied the oxidative metabolism of neutrophils after stimulation with recombinant GM-CSF (r.GM-CSF) and the concentrated conditioned medium of the UBC-5637 cell line (UBC-CM) showing CSF activity. It could be demonstrated that the r.GM-CSF, as well as the UBC-CM, induce an activation of the neutrophil respiratory burst without any cofactors such as f-MLP, PMA, or zymosan. In addition, we observed an increase of the response to those stimulants in the presence of either r.GM-CSF or UBC-CM. These effects were not endotoxin-induced, since stimulation persisted after addition of Polymyxin B, which is known to inhibit the action of endotoxins.     

Protective effect of taurine on respiratory burst activity of polymorphonuclear leukocytes in endotoxemia

Summary. The aim of this study was to evaluate the effect of endotoxin on PMN leukocyte respiratory burst activity by measuring G6PD, NADPH oxidase and XO activities in guinea pig. In addition, the possible protective role of taurine against endotoxin-mediated PMN leukocyte function was examined. All experiments were performed with four groups (control, taurine, endotoxemia, taurine plus endotoxin) of ten guinea pigs. After the endotoxin was administrated (4 mg/kg) both G6PD and NADPH oxidase activities were significantly reduced compared with the control group. NADPH oxidase activity returned to the control value and G6PD activity also increased but it did not reach the control value. However when taurine was administrated (300 mg/kg) the activity of NADPH oxidase reached the control value; furthermore, G6PD activity also increased but it could not reach to the control value. When taurine was administrated alone, no effect on these enzymes was observed. Following the endotoxin administration, the activity of XO considerably increased. When taurine was administrated together with endotoxine and alone, this activity decreased compared to control value in both conditions. These results indicate that the O₂ ^•− formation in PMN leukocytes after the endotoxin administration is ensured by the catalysis of XO due to the inhibited NADPH oxidase activity. It was observed that taurine has considerable anti-inflammatory and antioxidant effects. However, conflicting results were obtained when taurine was administrated alone or together with an oxidant agent.     

Inhibition of the neutrophil oxidative response to a chemotactic peptide by inhibitors of arachidonic acid oxygenation

N-formylmethionylphenylalanine stimulates a short burst of antimycin A-insensitive O₂ uptake, O₂^? production and hexosemonophosphate shunt oxidation of glucose by guinea pig peritoneal neutrophils. The stimulated oxidative metabolism, as well as release of lysosomal enzymes ± cytochalasin B, are inhibited by 5,8,11,14-eicosatetraynoic acid (ID₅₀ 1.5 × 10^?5 M). High concentrations of indomethacin inhibit the peptide-stimulated oxidations (ID₅₀ 1.6 × 10^?4 M) while acetylsalicylic acid (2.5 × 10^?3 M) does not. Digitonin-stimulated oxidative metabolism and enzyme release are not inhibited by 5,8,11,14-eicosatetraynoic acid or indomethacin at concentrations that depress effects of the N-formylated peptide.     

Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes

Oxidative metabolic burst of activated human polymorphonuclear leukocytes (PMN) is most commonly investigated in clinical practice by evaluating nitroblue tetrazolium (NBT) reduction at the single cell level. Reduced NBT precipitates where the redox reaction has taken place and can be visualized as PMN-associated dark blue granules of formazan in light microscopy. Although widely used and not technically demanding, this method remains subjective and labor intensive, especially when large numbers of samples need to be investigated. We developed a new flow cytometry technique in which PMN membrane was rendered fluorescent by a short incubation with fluorescein-conjugated Concanavalin A. PMN were then incubated with NBT and increasing doses of a suitable stimulus, such as phorbol myristate acetate (PMA). Formazan has a distinct peak of absorption at 520 nm that represents the peak of emission of fluorescein. As a consequence, formazan quenches the PMN-associated fluorescence. Data show that a dose-dependent reduction of fluorescence can be obtained using graded amounts of PMA in normal PMN cultures. PMN-associated fluorescence remains unchanged in control patients with chronic granulomatous (CGD) disease, a disorder characterized by a selective impairment of PMN oxidative metabolism. Electronic cell size increases upon PMA incubation in normal PMN, irrespective of the presence of NBT. Conversely, forward light scatter intensity decreases in the presence, but not in the absence, of NBT indicating that the phenomenon is due to the capacity of formazan to absorb/scatter the incident light. The present method for easily detecting NBT reducing activity at single cell level by flow cytometry makes use of commonly available, inexpensive reagents and standard instrumentation. It could become a useful test for clinical purposes.     

Structural studies of migration inhibitory activity induced during murine tumor allograft rejection.

Lymphocytes from mice rejecting a tumor allograft produce a soluble activity that inhibits the migration of murine macrophages. The present studies show that this activity is stable with 56 °C heating for 30 min, is inactivated by trypsin, has a molecular size of about 45,000, and has an isoelectric point of 5.0–5.5. In addition, it does not inhibit the migration of guinea pig macrophages. These results indicate that this lymphokine is similar in its physicochemical properties to murine, guinea pig and human migration inhibitory factors (MIF) studied previously, but they also suggest that there may be structural differences in the functionally active portions of MIF from different species.     

The Effect In vitro of High-Density Lipoprotein from Healthy and Infected Humans on the Oxidative Metabolism of Polymorphonuclear Leukocytes

We studied the effects in vitro of high-density lipoprotein from healthy (N-HDL) and from infected humans (AP-HDL) on the oxidative metabolism of human polymorphonuclear leukocytes (PMN). Products of the H₂O₂–MPO–halide system were monitored by luminol-enhanced chemiluminescence and superoxide anion formation was monitored by lucigenin-enhanced chemiluminescence during stimulation of human PMN with phorbol myristate acetate (PMA) or an opsonized stimulus (OS). The results showed that N-HDL and AP-HDL affect the oxidative metabolism of PMN in different ways. The posible role of this effect is discussed. © 1997 John Wiley & Sons, Ltd.     

Characterization of a factor produced by human alloreactive T lymphocyte clones, active on the eosinophils and precursors of erythroid strains

Alloreactive T-cell clones obtained from cells infiltrating a human rejected kidney graft were shown to produce upon specific antigenic stimulation with interleukin 2, a factor triggering the proliferation of an interleukin 3 dependent murine cell line. The lymphokine responsible for this activity was a monomeric glycoprotein (MW = 41,000) exhibiting on one hand chemotactic as well as activating properties on murine and human eosinophils respectively, and on the other hand a burst promoting activity. Its biochemical and biological features strongly suggest that this factor cannot be likened to any known human lymphokine.     

Immunological relatedness of human and porcine growth hormones.

The immunological relatedness of human and porcine growth hormones is examined by means of labelled human growth hormone and guinea pig antiserum. 1) Labelled human growth hormone is found in the precipitate after reaction with antiserum against porcine growth hormone. Parallel dilution curves are obtained with antisera against human and porcine growth hormones. 2) After addition of antiserum against porcine growth hormone, all the radioactivity is eluted from Sephadex G-100 with the void volume. 3) The addition of an excess of porcine hormone displaces labelled human growth hormone from antibodies against human growth hormone to the same extent as an excess of non-labelled human growth hormone does. 4) The standard radioimmunoprecipitation curves for porcine and human growth hormones obtained in the assay system for the human hormone are parallel in slope, provided that the human hormone and our preparation of the porcine hormone are introduced at a proportion of 1 to 560. 5) In a double diffusion test in agarose gel layers, with human and porcine growth hormones diffusing against guinea pig anti-porcine serum, cross reaction is observed. The conclusion is drawn that with guinea pig antisera, human and porcine growth hormones behave immunologically in a similar fashion. Labelled human growth hormone seems to have only such immunodeterminants as are also found in porcine growth hormone.