Selective priming of rate and duration of the respiratory burst of neutrophils by 1,2-diacyl and 1-O-alkyl-2-acyl diglycerides. Possible relation to effects on protein kinase C

Both 1,2-diacyl- and 1-O-alkyl-2-acyl-sn-glycerols are released during stimulation of human polymorphonuclear leukocytes (PMNL). 1,2-Diacylglycerols have received intense interest as intracellular "second messengers" due to their ability to activate protein kinase C (Ca2+ phospholipid-dependent enzyme). However, little is known about bioactivities of the alkylacylglycerols. This study compared the ability of 1,2-diacyl- and 1-O-alkyl-2-acylglycerols to modulate the respiratory burst of stimulated PMNL, a response which depends on the activation of an NADPH oxidase to generate bactericidal species of reduced oxygen. Direct stimulation by N-formyl-Met-Leu-Phe caused an abrupt release of H2O2 which ceased within 2.5 min. Preincubation with diacylglycerols (1-oleoyl-2-acetylglycerol,5-30 microM, and 1,2-dioctanoylglycerol,2-5 microM) caused a decrease in lag time, 3-fold increase in initial rate of H2O2 release, and marked prolongation of the response to N-formyl-Met-Leu-Phe (features characteristic of a priming effect). Preincubation with alkylacylglycerols (1-O-delta 9-octadecenyl-2-acetylglycerol, 5-30 microM, and 1-O-octyl-2-octanoylglycerol, 20-50 microM) primed initiation (shortened lag time and increased velocity) but, in contrast to diacylglycerols, did not alter duration of H2O2 release. While low concentrations of diacylglycerols (5-30 microM) primed PMNL, higher concentrations (greater than or equal to 70 microM) stimulated the cells directly. In contrast, higher (70-100 microM) concentrations of alkylacylglycerols did not prime the responses but, in fact, inhibited priming (especially of duration) induced by diacylglycerol. The high concentrations of alkylacylglycerol also inhibited direct stimulation induced by high concentrations of diacylglycerol. Direct stimulation by high concentrations of diacylglycerol probably involves activation of protein kinase C, whereas alkylacylglycerol was found to inhibit activation of protein kinase C by diacylglycerol in vitro. Thus, diacylglycerols are complete priming agonists, altering both rate and duration of the response. In contrast, alkylacylglycerols may have biphasic, concentration-related effects in modulation of functions of PMNL. At low concentrations, they may facilitate initiation of functional events; however, as their concentration increases, they may serve to terminate responses. The distinct priming effects of these diglycerides also reveal that priming can involve at least two distinct events: 1) initiation and 2) prolongation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of MHC-restricted rat T cells by cloned syngeneic thyrocytes

We have previously demonstrated that rat thyrocytes express MHC class II Ag (RT1.B&D) in response to IFN-gamma. To determine whether MHC class II-positive thyrocytes can be recognized by MHC-restricted T cells, we used our clone of rat thyroid cells (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) and known to express MHC class II Ag in response to recombinant rat IFN-gamma. CD4+ and CD8+ normal syngeneic Fisher rat spleen T cells were selected by flow cytometry and averaged greater than 96% purity. We demonstrated that irradiated MHC class II-positive but not class II-negative 1B-6 thyrocytes stimulated CD4+ T cells in a primary sensitization reaction over 4 days. In contrast, CD8+ T cells had no response in similar experiments. This stimulation of CD4+ T cells was dose dependent for 1B-6 thyrocytes and was abrogated by anti-rat MHC class II mAb (MRC OX-6). Autoreactive (Fisher) and alloreactive (Buffalo) T cell lines and isolated CD4+ T cells derived from these lines, which were developed against Fisher rat spleen cells, similarly recognized MHC class II Ag expressed on 1B-6 cells but had no detectable response to 1B-6 MHC class II-negative thyrocytes or MHC class II-positive human thyroid cells. The CD4+ T cell recognition of 1B-6 cells via MHC class II Ag supports our previous data with autologous human thyroid T cell co-cultures and is indicative of an autospecific role for thyrocytes in the development of autoimmune thyroiditis.     

Mechanism of copper transport from plasma to hepatocytes

The effects of plasma components on the kinetics of copper transport by rat hepatocytes were examined in an attempt to determine how copper is mobilized from plasma for uptake by the liver. Specific protein-facilitated transport was indicated by saturation kinetics, competition by related substrates, and similar kinetic parameters for uptake and efflux. For copper uptake, Km = 11 +/- 0.6 microM and Vmax = 2.7 +/- 0.6 nmol Cu/(min X mg protein). Zinc is a competitive inhibitor of copper uptake, and copper competes for zinc uptake. Copper efflux from preloaded cells is biphasic. The kinetic parameters for the initial rapid phase are similar to the parameters for uptake. Copper transport by hepatocytes is strictly passive. A variety of metabolic inhibitors have no effect on uptake and initial rates are solely dependent on extracellular-intracellular concentration gradients. Albumin markedly inhibits copper uptake by a substrate removal mechanism, and histidine facilitates albumin-inhibited copper uptake. The active species that delivers copper to hepatocytes under conditions of excess albumin and excess histidine is the His2Cu complex. Experiments with [3H]His2 64Cu showed that the transported species is free ionic copper. The kinetic parameters of copper transport by hepatocytes isolated from the brindled mouse model of Menkes' disease are normal. However, these cells show a decreased capacity to accumulate copper on prolonged incubation. An intracellular metabolic defect seems to be involved.     

Stimulation of hexose transport by human polymorphonuclear leukocytes: a possible role for protein kinase C

The protein C kinase activators 1-O-oleoyl, 2-O-acetylglycerol, 12-O-tetradecanoyl phorbol-13-acetate, and mezerein, stimulated deoxyglucose uptake in human neutrophils. The responses were stimulus specific since no effect was noted with the diether analogues 1-O-hexadecyl-2-O-ethylglycerol, 1-O-palmitoyl-2-O-acetyl or 1-O-palmitoyl-3-O-acetyl diesters of propanediol, or with 1,2-diolein. Stimulation of deoxyglucose uptake had the characteristics of carrier facilitated hexose transport. Stimulated uptake of deoxy-glucose was inhibited by trifluoperazine (10-30 microM). Activation of protein kinase C therefore appears to trigger events involved in hexose transport.     

The hepatic response to Ca2+ is inhibited by Mg2+ and enhanced by phenylephrine or ouabain

Net hepatic Ca2+ efflux, K+ uptake and glycogen breakdown in response to the alpha 1-adrenergic agonist phenylephrine were studied. Rat livers were perfused with CO2/bicarbonate-buffered solutions containing 10 microM Ca2+ and different amounts of Mg2+. K+-free medium and/or ouabain were used to block (Na+ + K+)-ATPase-dependent K+ uptake. In some experiments a sharp increase in extracellular Ca2+ concentrations was produced by infusing CaCl2 into the medium entering the liver. Perfusion with K+-free medium and ouabain enhanced the phenylephrine-induced Ca2+ efflux and diminished the glycogenolytic response, indicating a dissociation of Ca2+ release and glycogenolysis. Exogenous Ca2+ had practically no effect if livers were perfused with regular medium containing 1.2 mM Mg2+. In the presence of phenylephrine and if extracellular Mg2+ concentrations were lowered by omitting Mg2+ from the medium or by preperfusion with EGTA, exogenous Ca2+ was glycogenolytically effective and also produced a transient K+ uptake. Increased extracellular concentrations of Mg2+ inhibited the effects of exogenous Ca2+. In the presence of phenylephrine, higher concentrations of Mg2+ were needed than in the absence of alpha 1-adrenergic agonist to achieve a similar degree of inhibition. In one respect ouabain effects were comparable to those of phenylephrine: the glycoside also increased the metabolic response to exogenous Ca2+ and diminished the sensitivity towards Mg2+. Phenylephrine and ouabain may both enhance the permeability of plasma membranes for Ca2+.     

Regulation of the oxidative response of human granulocytes to chemoattractants. No evidence for stimulated traffic of redox enzymes between endo and plasma membranes

Experiments were performed to probe the role of exocytotic and endocytotic processes in the regulation of the human granulocyte O-2-generating system. Analytical subcellular fractionation studies indicated that 25-30% of the total cellular b-cytochrome and 8-10% of the flavin co-sedimented with plasma membrane markers, irrespective of stimulation of the cells by the chemoattractants N-formyl-Met-Leu-Phe (FMLP) or C5a. Phorbol myristate acetate stimulation resulted in significant translocation of b-cytochrome but not flavin from the specific granule/Golgi to the plasma membrane-enriched fractions. These results indicated that approximately 3.1 X 10(5) flavin and 0.8-1 X 10(6) b-cytochrome molecules are present in the plasma membrane of an isolated unstimulated human granulocyte and that these levels are invariant upon stimulation with chemoattractants. Maximal instantaneous rates of O-2 generation by cells in these preparations, however, were equivalent for all the stimuli. Since stimulation of granulocytes by phorbol myristate acetate, FMLP, or C5a results in exocytosis and/or endocytosis, then the role of these processes in regulating stimulated O-2 production by controlling the content of plasma membrane redox enzymes is questionable. This conclusion was supported by observations made with cytoplasts, which do not have an intracellular reserve of granules. Cytoplasts prepared from granulocytes produced O-2 at equivalent rates as their parent cells on a per unit surface area basis. These results suggest: 1) that stimulation of granulocytes with chemotactic peptides leads to full generation of O-2 at the cell surface without exocytotic recruitment of additional b-cytochrome and flavoprotein from the cytoplasmic compartment; 2) that these redox enzymes are not internalized along with chemoattractant receptors; and 3) that traffic of these redox enzymes between endo- and plasma membranes is not involved in the regulation of O-2 production in suspensions of human granulocytes stimulated by chemoattractants.     

Immunoregulation in disseminated murine histoplasmosis: disturbances in the production of interleukins 1 and 2

Production of IL 1 and IL 2 by splenocytes from C57BL/6 mice was measured at wk 1, 3, 8, and 14 after i.v. inoculation with 6 X 10(5) Histoplasma capsulatum (Hc) yeasts. As compared with age-matched controls, IL 1 production by splenocytes from Hc-infected mice was reduced severely at wk 1 and 3 of infection, greater than normal at wk 8, and within normal range at wk 14. IL 2 production was also reduced at wk 1 and 3 of infection; it was normal at wk 8 and was elevated at wk 14. Indomethacin and catalase failed to restore IL 1 production by splenocytes from infected mice, and exogenous IL 1 did not augment IL 2 production by these cells. A factor capable of suppressing the activity of IL 2 was detected in supernatants of concanavalin A-stimulated splenocytes from infected animals at wk 1 and 3 of infection, respectively. No factor capable of suppressing IL 1 activity was detected. Thus, the deficits of cell-mediated immunity in mice with systemic Hc infection may derive, in part, from impaired amplification of the immune response consequent to abnormal generation of IL 1 and IL 2.     

Structure of complex flagellar filaments in Rhizobium meliloti

The complex flagella of Rhizobium meliloti 2011 and MVII-1 were analyzed with regard to serology, fine structure, subunits, and amino acid composition. The serological identities of flagellar filaments of the two strains were demonstrated by double immunodiffusion with antiflagellin antiserum. The filaments had a diameter of 16 nm. Their morphology was dominated by the prominent undulations of an external three-start helix running at a 10-nm axial distance and at an angle of 32 degrees. Faint nearly axial striations indicated the presence of a tubular core of a different helical order. The complex filaments consisted of 40,000-dalton flagellin monomers. Typically, the amino acid composition was 3 to 4% higher in nonpolar residues and 5 to 7% lower in aspartic and glutamic acids (and their amides) than that of plain flagellar proteins. There were no immunochemical relationships among Pseudomonas rhodos, Rhizobium lupini, and R. meliloti complex flagella, suggesting that the latter represent a new class.     

Population Dynamics of Heterodera glycines Life Stages on Soybean

Population fluctuations of Heterodera glycines differ in fields with high and low initial population densities. In a field with low initial numbers of nematodes, the numbers of cysts and eggs in soil remained low through 100 days from planting then increased during the remainder of the growing season. In a field with high initial nematode populations, numbers increased at 30 days, decreased to low numbers at 100 days, and then resurged to maximum populations at harvest. Numbers of juveniles were greatest at 100 days in the low initial population density field and at planting in the high initial population density field. The initial numbers of eggs in the soil gave the best correlation to soil and root nematode populations 15 and 30 days later. Juveniles in the soil at planting gave the largest correlation coefficients with nematode populations in the roots at 15 days in the field with the low initial population density. Eggs and juveniles in the soil at harvest were poorly related to numbers that overwintered.