Enhancement by growth hormone of phorbol diester-stimulated respiratory burst in human polymorphonuclear leukocytes

The oxidative metabolic burst of mononuclear and polymorphonuclear phagocytes can be stimulated to produce free oxygen radicals. Several substances can enhance this respiratory burst activity by a priming action: recently growth hormone (rat and porcine) was demonstrated to act as a priming agent on rat peritoneal and on porcine alveolar macrophages. In our study we wanted to verify whether also human GH had a similar priming action on homologous cells, in particular on polymorphonuclear leukocytes. To determine the oxidative activity of polymorphonuclear leukocytes, after stimulation with phorbol myristate acetate, a flow-cytometric assay was employed which registered the intracellular formation of highly fluorescent products as indicators for the intracellular formation of hydrogen peroxide. The incubation of phorbol myristate acetate-stimulated polymorphonuclear leukocytes with GH resulted in a time-dependent and dose-dependent increase in fluorescence, thus demonstrating that human GH enhances in vitro the oxidative metabolic burst of these cells. The action of GH appeared to be significant after 30 min of incubation, was maximal at 60 min, and decreased after 90 min. After one hour of incubation, the first significant variation of fluorescence appeared with GH at a concentration of 50 micrograms/l. The maximum effect was seen at 100 micrograms/l with no further increase. Specificity of GH action was demonstrated by the inhibition of its effect by the addition of monoclonal antibodies to GH.     

Chemoattractant-mediated stimulation of the respiratory burst in human polymorphonuclear leukocytes may require appearance of protein kinase activity in the cells' particulate fraction

Low doses of aliphatic alcohols produce divergent effects on the function of chemoattractant receptors on human polymorphonuclear leukocytes (PMNs) since they enhance chemotaxis but inhibit stimulation of superoxide production by chemoattractants. As such, alcohols can provide useful pharmacologic tools to probe the mechanisms of stimulus- response coupling in leukocytes. A role for protein kinase C has been implicated in the activation of the respiratory burst in PMNs. Although the vast majority of this enzyme activity is located in the cytosolic fraction of unactivated PMNs, protein kinase C activity appears in the particulate fraction of the cells when they are stimulated to produce superoxide by either chemoattractants or by phorbol myristate acetate (PMA). Doses of the alcohols that selectively inhibited stimulation of superoxide production by chemoattractants also inhibited the appearance of protein kinase C activity as well as an undefined protein kinase activity in the particulate fraction of the cells. In contrast, the alcohols did not affect either the ability of PMA to stimulate the production of superoxide in PMNs nor the appearance of protein kinase activity in the cells' particulate fraction. PMA is known to bind and activate protein kinase C directly, thus bypassing receptor-mediated events. These data suggest that alcohols inhibit the stimulation of the respiratory burst by chemoattractants in PMNs by blocking the ability of receptor occupancy to induce the appearance of protein kinase activity in particulate fractions. These results moreover suggest that the appearance of protein kinase activity in the particulate fraction may be required for activation of the respiratory burst in PMNs.     

Extracellular cytotoxicity by phagocytosing polymorphonuclear neutrophilic leukocytes: enhancement by a chemotactic stimulus

This study investigated the influence of a chemotactic stimulus on the extracellular cytotoxicity mediated by phagocytosing polymorphonuclear neutrophilic leukocytes (PMN). We used N-formyl-methionyl-leucyl-phenylalanine (FMLP) as chemotactic peptide, opsonized zymosan as phagocytosable particle, and ox red blood cells (ORBC) as extracellular bystander targets. Phagocytosing PMN were found to kill ORBC efficiently, as determined by the 51Cr-release assay. FMLP, at the concentration of 100 nM, significantly enhanced the target cell lysis. PMN from two patients with chronic granulomatous disease and normal PMN plus catalase or free radical scavengers (mannitol, benzoate, histidine) were completely devoid of cytolytic activity both in the presence and in the absence of FMLP. The results indicate that the target cell lysis by phagocytosing PMN as well as the chemotactic peptide-related amplification of the lysis itself depend on the expression of the PMN oxidative cytotoxic potential. A similar response to a chemotactic stimulus in vivo could provide a mechanism for regulating PMN-dependent cytotoxic and inflammatory processes.     

Demonstration of specific C5a receptor on intact human polymorphonuclear leukocytes.

Human C5a, a complement-derived anaphylatoxin, is a potent mediator of human leukocyte chemotaxis. Using a homogeneous preparation of C5a that was 125I-labeled, we have demonstrated the presence of a specific cellular receptor for this glycoprotein on intact human polymorphonuclear leukocytes. Cellular uptake of the radiolabeled ligand occurred rapidly and the rate of dissociation was extremely slow. Cellular binding was saturable with respect to 125I-labeled C5a, and half-saturation occurred at a concentration of 3-7 X 10(-9) M. The number of C5a binding sites per cell was estimated as 1-3 X 10(5). The ligand (C5a) displays specific structural features that are required for binding because analogs of C5a such as C5ades Arg or a yeast carboxypeptidase-digested C5a derivative C5a-(I-69) inhibited the binding but C3a anaphylatoxin, which resembles C5a chemically, did not. Both C5a-mediated leukocyte chemotaxis and C5a-induced lysosomal enzyme release from cytochalasin B-treated cells closely paralleled uptake of the ligand, clearly indicating that it is a receptor-C5a interaction that leads to stimulation of these cellular responses.     

Oxidative inactivation of leukotriene C4 by stimulated human polymorphonuclear leukocytes

Leukotriene C₄ (LTC₄) was metabolized by human polymorphonuclear leukocytes (PMNs) stimulated with phorbol myristate acetate (PMA) into three sets of products. These products differed in mobility on reverse-phase high-performance liquid chromatography (RP HPLC) from LTC₄ and also from leukotriene D₄ (LTD₄) and leukotriene E₄ (LTE₄), the sequential products of peptide cleavage of LTC₄. Products I, II, and III were eluted as doublets with an average retention time for each doublet of 7.5 ± 0.3, 10.5 ± 0.6, and 16.3 ± 1.1 min (mean ± SD), respectively, as compared with 13.8 min for LTC₄. Doublet I material was biologically inactive and showed <5% of the immunoreactivity of LTC₄, doublet II material had 1% of the spasmogenic activity of LTC₄ on the guinea pig ileum and was equally immunoreactive, and doublet III material was neither biologically active nor immunoreactive. When [14,15-³H]LTC₄ and [³⁵S]LTC₄ were metabolized, all three doublet products retained the ³H label, whereas only the doublet I and doublet II products retained the ³⁵S label. The UV absorbance spectra of the three sets of metabolites were as follows: doublet I, maximum at 280 nm with shoulders at about 270 and 290 nm; doublet II, maximum at 284.5 nm with shoulders at about 275 and 295 nm; and doublet III, maximum at 269 nm with shoulders at about 259 and 279 nm. The metabolism of LTC₄ to the three classes of functionally inactive products by stimulated PMNs was completely blocked by catalase and azide, indicating a requirement for H₂O₂ and myeloperoxidase. When hypochlorous acid (HOCl)—considered to be a natural product of the interaction of myeloperoxidase, H₂O₂, and chloride ion—was formed chemically and allowed to react with LTC₄, the resulting products were indistinguishable by UV and HPLC analyses from the doublet II and doublet III metabolites of LTC₄. The doublet II products were identified as the two diastereoisomeric sulfoxides of LTC₄ by comparison with synthetic reference compounds. The doublet III products were shown to be identical with synthetic samples of (5S, 12S)- and (5S, 12R)-6-trans-LTB₄. The formation of two diastereoisomeric LTC₄ sulfoxides and 6-trans-LTB₄ can be explained in terms of an S-chlorosulfonium ion as the initial reactive intermediate, which subsequently undergoes conversion to product II by hydrolysis and product III by carbocation formation.     

Increased polymorphonuclear leukocyte respiratory burst function in type 2 diabetes

The predisposition to infection and chronic inflammation in diabetes may in part be related to the effects of hyperglycemia or other metabolic abnormality on polymorphonuclear leukocytes (PMN). We evaluated oxidative respiratory burst activity (superoxide production) in non-stimulated and stimulated PMN from 70 stable type 2 Hispanic diabetic patients, as compared to 70 healthy Hispanic individuals without diabetes. The influences of protein kinase C (PKC) inhibitors and certain antibiotics on superoxide production were examined. Both resting and stimulated (PMA, zymosan) PMN from diabetic individuals produced more superoxide than PMN from controls. Inhibitors of PKC, a possible mediator of the augmented respiratory burst activity, decreased superoxide production in all (resting and stimulated) diabetic and control PMN. Azithromycin, which is markedly concentrated by PMN, profoundly inhibited superoxide generation in all groups of diabetic and control cells. PMN from Hispanic diabetic patients produced greater quantities of superoxide than non-diabetic controls. This increased oxidative respiratory burst activity may predispose to infection and chronic inflammation in diabetes. PKC inhibitors and azithromycin inhibited this respiratory burst response. The possible role of PKC (especially PKC beta) as the mediator of this augmented respiratory burst response requires further evaluation, and may lead to therapeutic studies with appropriate inhibitors.     

Enhancement of hexose uptake in human polymorphonuclear leukocytes by activated complement component C5a

The polymorphonuclear leukocyte (PMNL) depends on glucose as a source of energy for motility, chemotaxis, phagocytosis, and bactericidal activity. Activated complement (C5a) at low concentrations stimulates carrier-mediated carbohydrate transport in PMNLs as measured by the uptake of 2-deoxy-D-[(3)H]glucose. Human PMNLs were preincubated at 37 degrees C for 15 min with zymosan-activated human serum or various purified preparations of human C5a. A concentration-dependent increase in deoxyglucose transport (>700% of control) into PMNLs occurred with all test substances. Reaction was linear for 30 min, and uptake of deoxyglucose followed saturation kinetics. C5a caused a decrease in the K(m) for deoxyglucose, from 0.53 to 0.11 mM, without altering the V(max) (44 nmol/30 min per 5 x 10(6) PMNLs in control and 46.6 with C5a). The optimal concentration of C5a for enhanced carrier-mediated transport of deoxyglucose was similar to that which promoted optimal chemotaxis. Activated serum from C5-deficient mice had little or no effect on deoxyglucose transport whereas that from normal syngeneic mice enhanced deoxyglucose transport. C5a did not enhance deoxyglucose transport into isolated erythrocytes, platelets, or lymphocytes. The deoxyglucose within the cell was primarily in the phosphorylated form, and hexokinase activity was not increased in PMNLs stimulated with C5a, indicating that hexokinase was not rate limiting and that enhanced transport was the mechanism of the C5a activity. Insulin at physiologic concentration (10 ng/ml) had no effect on deoxyglucose transport in PMNL and did not act as a competitive inhibitor of C5a. This insulin-like bioactivity could be detected with the amount of C5a that would be present after activation of 0.1-0.5% of the C5 in 1 ml of serum. This suggests that uptake of [(3)H]deoxyglucose by PMNLs might serve as a highly sensitive test for activation of the fifth component of complement.     

Cytoplasts made from human blood polymorphonuclear leukocytes with or without heat: preservation of both motile function and respiratory burst oxidase activity.

Anucleate fragments (cytoplasts) from polymorphonuclear leukocytes (PMN) are simplified systems that can be used to elucidate specific pathways by which cell function is altered. PMN cytoplasts in current use are defective either in activatable respiratory burst oxidase activity or in motile function. By centrifugation of PMN on discontinuous gradients of Ficoll without cytochalasin B, we have created granule-poor cytoplasts in which both these capacities are preserved. Specifically, they generate superoxide anion (O2-.) and reduce nitroblue tetrazolium dye on appropriate stimulation; they respond chemotactically to erythrocytes destroyed by laser microirradiation or to the specific chemoattractants fMet-Leu-Phe (10 nM) and C5a (zymosan-activated serum); and they ingest and kill staphylococci. We can improve the yield of these fragments progressively by preheating (45 degrees C) the cells in suspension for increasing periods of time, but those treatments are attended by a decreasing percentage of cytoplasts with activatable oxidase activity, and a progressive inability of the cytoplasts to ingest and to kill staphylococci. These easily made and multipotent cytoplasts readily lend themselves to studies of PMN physiology.     

Inhibitory effect of somatostatin on cAMP accumulation and calcitonin secretion in C-cells: involvement of pertussis toxin-sensitive G-proteins.

The effect of somatostatin on cAMP accumulation and calcitonin secretion in C-cells of the rat medullary thyroid carcinoma cell line rMTC 6-23 was investigated. Intracellular cAMP accumulation as well as calcitonin secretion could be dose-dependently stimulated by rat growth hormone releasing factor (rGRF). The long-acting somatostatin analogue octreotide inhibited rGRF-stimulated cAMP accumulation and calcitonin secretion dose dependently but failed to block 8-bromo-cAMP-stimulated calcitonin secretion. The inhibitory effect of octreotide on rGRF-induced calcitonin secretion was partially abolished by pretreating the cells with pertussis toxin. The octreotide effect was not due to changes in the degradation of cAMP, as it was similarly seen in the presence of isobutylmethylxanthine. Thus we conclude that pertussis toxin-sensitive G-proteins are involved in the cAMP-mediated regulation of calcitonin secretion in C-cells.     

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex.

We have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a Kd of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the alpha and beta subunits of Gi, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm our earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.     

Independent stimulation of membrane potential changes and the oxidative metabolic burst in polymorphonuclear leukocytes

Early events of stimulus-response coupling in polymorphonuclear leukocytes (PMNL) reportedly include membrane depolarization as a necessary antecedent to oxidative responses. However, depolarization by nonspecific means (ionophores) is insufficient to elicit an oxidative burst. This apparent conflict might be related to whether depolarization is due to membrane receptor-mediated stimulation of PMNL. We used two fluorescent probes and dual laser flow cytometry to monitor both membrane potential and oxidative product formation in individual PMNL, following stimulation by phorbol myristate acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP). Dipentylindocarbocyanine [di-I-C5(3)] is one of a family of dyes that partition between cells and aqueous media as a function of transmembrane potential. The dye appeared stable in the presence of PMNL oxidative products. Oxidation of intracellularly trapped, nonfluorescent dichlorofluorescin (DCFH) to fluorescent dichlorofluorescein (DCF) provided a quantitative assessment of oxidative metabolism (H2O2 production) of stimulated PMNL. Incubating PMNL with both fluorochromes resulted in stable red [di-l-C5(3)] and green (DCF) fluorescence when examined on a Cytofluorograph. Upon stimulation by 0.03 to 0.1 nmol/L PMA, PMNL showed a unimodal apparent depolarization (decrease in di-l-C5(3) fluorescence). Oxidative activity (increased DCF fluorescence) was first seen at a concentration of PMA of 0.17 nmol/L, higher than that required to elicit depolarization. This oxidative burst appeared as a dose-dependent, graded response. Thus, receptor-mediated membrane depolarization, although antecedent to the onset of the oxidative burst, was not in itself sufficient to trigger the oxidative metabolic response. When PMNL were isolated by centrifugation through Ficoll-metrizoate, fMLP caused an apparent depolarization of a variable subpopulation of the cells. However, such purified PMNL appeared relatively unstable and often depolarized spontaneously. PMNL studied without centrifugation through Ficoll-metrizoate were stable. Moreover, fMLP stimulation of such cells did not cause membrane depolarization but did stimulate a two- to six-fold increase in DCFH oxidation. Apparently, membrane depolarization may antecede oxidative responses in PMNL, but appears to depend upon the method of cell preparation and the nature of the stimulus.     

Conjugated bile acids promote ERK1/2 and AKT activation via a pertussis toxin-sensitive mechanism in murine and human hepatocytes

Several studies have argued that G-protein-coupled receptors (GPCR) have the capacity to promote activation of receptor tyrosine kinases. The current studies were performed to examine the regulation of the extracellular regulated kinase (ERK)1/2 and AKT pathways by conjugated and unconjugated bile acids in primary hepatocytes. Deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), taurocholic acid (TCA), glycocholic acid (GCA), and tauroursodeoxycholic acid (TUDCA) all activated ERK1/2 in primary rat hepatocytes that was abolished by inhibition of ERBB1, and significantly reduced by ROS quenching agents. Bile acid-induced AKT activation was blunted by preventing ERBB1 activation and ROS generation. Treatment of rat hepatocytes with pertussis toxin (PTX) did not alter ERK1/2 and AKT activation induced by DCA or CDCA but abolished pathway activations by conjugated bile acids. Similar data to those with PTX were obtained when a dominant negative form of G(i1alpha) was overexpressed. Treatment of rat hepatocytes with TDCA and TCA promoted guanosine triphosphate (GTP) loading of G(i1alpha), G(i2alpha), and G(i3alpha) in vitro. Treatment of rat hepatocytes with PTX abolished TDCA-induced tyrosine phosphorylation of ERBB1. Similar findings to those in rat hepatocytes were also obtained in primary mouse and human hepatocytes, but not in established rodent or human hepatoma cell lines. In conclusion, collectively our findings demonstrate that unconjugated bile acids activate hepatocyte receptor tyrosine kinases and intracellular signaling pathways in a ROS-dependent manner. In contrast, conjugated bile acids primarily activate receptor tyrosine kinases and intracellular signaling pathways in a GPCR (G(ialpha))-dependent and ROS-dependent manner.     

Monoclonal Lym-1 antibody-dependent lysis of B-lymphoblastoid tumor targets by human complement and cytokinine-exposed mononuclear and neutrophilic polymorphonuclear leukocytes

Lym-1 is a murine IgG2a monoclonal antibody that recognizes a polymorphic variant of HLA-DR antigens on malignant B cells, with minimal cross-reactivity with normal tissues. Because it can be safely administered in vivo, a detailed knowledge of its ability to recruit and trigger the antitumor immune effector systems is required to optimize potential serotherapeutic approaches in B-lymphoma patients. By using Raji cells as a model of B-lymphoma targets, we found that Lym- 1 activates complement-mediated lysis efficiently. Moreover, Lym-1 was capable of triggering the antibody-dependent cellular cytolysis (ADCC) by peripheral blood mononuclear cells (MNCs). On the contrary, it failed to trigger neutrophilic polymorphonuclear leukocyte (PMN)- mediated ADCC activity. In an attempt to enhance Lym-1 ADCC by MNCs and PMNs, nine biologic response modifiers were tested. MNC-mediated Lym-1 ADCC was significantly stimulated by interleukin-2 (IL-2) and unaffected by other mediators, including gamma-interferon (gamma-IFN), tumor necrosis factor a (TNFalpha), and granulocyte-macrophage colony- stimulating factor (GM-CSF). On the other hand, PMN-mediated Lym-1 ADCC was induced or significantly augmented by various cytokines, such as GM- CSF, TNFalpha, and gamma-IFN, and chemotaxins, such as formyl peptides (FMLP), complement fragment C5a, and IL-8. Both MNC- and PMN-mediated ADCC was unaffected by granulocyte colony-stimulating factor (G- CSF) and insulin-like growth factor-1 (IGF-1). Finally, only GM-CSF and TNFalpha augmented the number of PMNs actually engaged in the binding of Raji target cells. The findings presented here, in particular those showing stimulatory activity of biologic response modifiers, may inspire new attempts for developing Lym-1 antibody-based approaches to the therapy of B lymphomas.     

Regulatory mechanisms of a chemoattractant receptor on human polymorphonuclear leukocytes

The oligopeptide chemotactic factor receptor in leukocyte membranes exists in two affinity states that are in part interconvertible. Convertibility is regulated by guanine nucleotides, which suggests that a nucleotide regulatory unit allosterically modifies receptor affinity and participates in its transduction mechanisms. Approximately one-third of the high-affinity receptors in polymorphonuclear leukocyte (PMN) membranes are not subject to guanine nucleotide regulation. This fraction can be increased by agonist preincubation and could represent an intermediate form of the receptor prior to signal transduction and/or internalization. Pharmacologic manipulation of viable PMNs demonstrates that the affinity and functional activity of the chemoattractant receptor can be altered divergently by aliphatic alcohols and polyene antibiotics. The alcohols raise the receptors' affinity, enhance chemotaxis, but markedly depress chemoattractant-induced secretory mechanisms. In contrast, polyene antibiotics lower the receptors' affinity, depress chemotaxis, but enhance specific granule secretion. Thus, a relationship exists between the chemoattractant receptors' affinity and their ability to transduce signals for either chemotaxis or secretion.     

Sickle erythrocytes adhere to polymorphonuclear neutrophils and activate the neutrophil respiratory burst

The vasoocclusive process in patients with sickle cell disease (SCD) is complex and involves interactions among sickle erythrocytes (SS-RBC), vascular endothelium, and plasma and cellular components. The role of neutrophils (PMN) in vasoocclusion has not been examined. Patients with SCD appear to have chronically activated PMN. Because the first step in PMN activation is particle recognition, we explored whether normal PMN recognize SS-RBC and whether this recognition results in PMN monolayers, significantly more SS-RBC adhered to the PMN than did normal erythrocytes (AA-RBC; P < .001). Preincubation of erythrocytes with autologous plasma significantly increased the adherence of SS-RBC to PMN but had no effect on AA-RBC (P < .001). When adhesion of density fractionated SS-RBC was performed, dense SS-RBC showed greater adherence to the PMN monolayers than did light SS-RBC (P < .001). To determine mechanisms of this adhesion, IgG and Arg-Gly-Asp-Ser (RGDS) receptor sites on PMN were saturated. IgG inhibited adherence of dense SS-RBC, whereas RGDS inhibited adherence in both fractions, although to a greater extent in the light fraction. We measured SS-RBC activation of PMN by incubating SS-RBC with 2', 7'-Dichloro-fluroescin Diacetate (DCF)-labeled PMN. Incubation of PMN with SS-RBC resulted in a significant increase in fluorescence compared to AA-RBC. We show here that PMN recognize SS-RBC through multiple mechanisms and that this recognition results in activation of PMN. These findings contribute to the understanding of vasoocclusive crisis in patients with SCD and may have therapeutic implications.     

Opsonized-zymosan induces a respiratory burst in human blood platelets

Opsonized-zymosan-stimulated polymorphonuclear cells show a cyanide-insensitive oxygen consumption. We have investigated whether opsonized-zymosan could induce similar metabolic change in human blood platelets. Preparation of intact human blood platelets, obtained by separation through a Ficoll layer (23% w/v) were challenged with opsonized-zymosan. The polymorphonuclear cell contamination was less than 1/10(8) platelets. The opsonized-zymosan-stimulated platelets showed an increase of oxygen consumption. The mean of oxygen burst measured by a polarographic method with a Clark electrode was 11 nmole/10(9) platelets/min (S.E.M. 4; n = 15). The duration of the burst was 2 min. Unstimulated platelets did not show the oxygen burst. The inhibitors of respiratory chain and prostaglandin synthesis completely abolished the oxygen consumption by opsonized-zymosan-stimulated platelets. The simultaneous addition of NADH (1 mM) and opsonized-zymosan induced a burst of oxygen consumption, which occurred after a variable lag phase (10-12 min) from the stimulation, also in the presence of inhibitors. This burst, which lasted about 1 min, amounted to 10 nmole/10(9) platelets/min (S.E.M. 2; n = 15) and it was higher in the presence of NAN3, a catalase inhibitor. Zymosan treated with hydrazine or heated plasma (56 degrees C) did not cause increased oxygen consumption. Inulin or inulin-treated serum did not stimulate platelets. In these experimental conditions some NADH disappeared, as shown by isotachophoresis. The results demonstrated that an immunological stimulus may activate a membrane-linked cyanide-insensitive oxygen metabolizing system.     

Functional analysis of the marginating pool of human polymorphonuclear leukocytes

The intravascular pool of human polymorphonuclear leukocytes (PMN) is composed of one compartment which is circulating and another that is marginated to the vascular endothelium. Administration of B-adrenergic agonists leads to a rapid demargination with an increase in the circulating PMN pool. The marginating PMN has previously been stated to represent an older PMN based on a higher cytochemical alkaline phosphatase activity. With the understanding that circulating PMN are heterogeneous with respect to function and size we undertook the present study to evaluate the contribution of the marginating PMN to functional and volume-dependent heterogeneity. We found that PMN isolated 7 min after epinephrine administration, presumably enriched by marginating PMN, were not different in volume, biochemically measured alkaline phosphatase activity, stimulated superoxide anion release, degranulation, or phagocytosis. These data suggest that the circulating and marginating pools of PMN are interchangeable and that the marginating PMN are not enriched by a particular subpopulation of PMN.     

Superoxide anion generation of human polymorphonuclear leukocytes in urine]

In order to clarify the role of polymorphonuclear leukocytes (PMNs) in the defence mechanism of the urinary tract, we investigated the superoxide anion (O2-) generation of PMNs in the urine. We tested urines mixing concentrated urine with diluted ones in various ratios. Peripheral blood derived PMNs were stimulated in tested urines or phosphate buffer solution (1M, pH 7.0) with phorbol-myristate acetate and O2- generation of PMNs was measured by means of chemiluminescence (CL) method. The CL values of PMNs in tested urines were 1/30 that of phosphate buffer solution. However, PMNs generated O2- at maximum levels when the urine osmolarity was 300-400 mOsm/kg. Furthermore, PMNs did not generate O2- when the urine osmolarity was more than 600 mOsm/kg, or less than 200 mOsm/kg. We also investigated the influence of urea and creatinine on O2- generation of PMNs. The O2- generation of PMNs decreased when the urea concentration rose. However, there was no influence of creatinine on O2- generation of PMNs. From these results, it was elucidated that PMNs generated O2- when the urine osmolarity was 300-400 mOsm/kg. Therefore, urine osmolarity of the patients with urinary tract infection should be kept at 300-400 mOsm/kg, and diuresis might be important for the patients with urinary tract infection.     

Interleukin 3 activates human blood basophils via high-affinity binding sites

Pure populations of human basophilic granulocytes were obtained from chronic myeloid leukemia (CML) blood by negative selection using a mixture of monoclonal antibodies and complement. 125I-radiolabeled recombinant human interleukin 3 (rhIL-3) bound to purified basophils in a specific manner. Quantitative binding studies and Scatchard plot analyses performed on samples from two donors revealed the presence of a single class of high-affinity IL-3 binding sites (500 and 2100 sites per cell; dissociation constant at equilibrium, 230 and 160 pmol/liter, respectively). Purified CML basophils maintained in suspension in the presence of rhIL-3 (100 units/ml) incorporated up to 12 times more [3H]thymidine than basophils in control cultures. Furthermore, after preincubation in vitro with rhIL-3 (100 units/ml) for 30 min, normal blood basophils released 2- to 3-fold more histamine than basophils pretreated with control medium when exposed to various concentrations of an anti-IgE antibody. Together, these results show that rhIL-3 binds to a specific receptor on blood basophils and is a regulator of basophil function.     

Identification of the pertussis toxin-sensitive G proteins in platelets, megakaryocytes, and human erythroleukemia cells

Guanine nucleotide-binding regulatory proteins, or G proteins, mediate the interaction of agonist receptors on the platelet surface with phospholipase C and adenylyl cyclase. To better understand this process, we have used several approaches to identify which G proteins are present in platelets, normal human megakaryocytes, and human erythroleukemia (HEL) cells, a leukemic cell line with megakaryocytic features. Because platelet and HEL cell responses to thrombin are inhibited by pertussis toxin, we have focused upon the members of the Gi family, whose alpha subunits can be ADP-ribosylated by that toxin. Western blots with antisera specific for Gi alpha demonstrated the presence in both platelets and HEL cells of the three best-described forms of this protein: Gi alpha 1, Gi alpha 2, and Gi alpha 3. Based upon immunoprecipitation studies with [35S]-methionine-labeled HEL cells, their relative abundance appears to be Gi alpha 2 much greater than Gi alpha 3 greater than Gi alpha 1. A HEL cell cDNA library screened with the Gi alpha antisera produced clones encoding Gi alpha 2 and Gi alpha 3 that had sequences similar to those reported from other sources. Gi alpha-specific probes created from these cDNA clones confirmed the presence of mRNA encoding Gi alpha 2 and Gi alpha 3 in both platelets (by Northern blotting) and megakaryocytes (by in situ hybridization). Thus the pertussis toxin substrates that have previously been detected in platelets and HEL cells are shown to be members of the Gi alpha family, all of which are candidates for interaction with receptors for thrombin and other agonists.