Priming of the respiratory burst of neutrophils by diacylglycerol. Independence from activation or translocation of protein kinase C.

Preincubation of neutrophils with certain agonists may "prime" the cells to cause increased responses to a second stimulus ("primed stimulation"). We used two approaches to examine the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in priming and stimulation by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and N-formyl-Met-Leu-Phe (fMLP): inhibition of protein kinase C by 1-(5-isoquinolinesulfonyl)-piperazine (C-I) and measurement of protein kinase C translocation induced by priming and stimulatory concentrations of OAG. C-I had little effect on stimulation or primed stimulation by fMLP, suggesting that fMLP invokes events independent of protein kinase C. C-I equally inhibited stimulation and primed stimulation by PMA. Direct stimulation by OAG was inhibited, but priming and primed stimulation by OAG was unaltered by C-I. OAG concentrations greater than or equal to 100 microM caused translocation of protein kinase C, in correlation with direct stimulation of the respiratory burst. Lower OAG concentrations (10-30 microM) primed to stimulation by fMLP and, conversely, stimulated neutrophils primed with fMLP, yet did not cause translocation of protein kinase C. The data are compatible with previous assumptions that PMA and OAG directly stimulate polymorphonuclear neutrophil leukocytes by translocation and activation of protein kinase C. However, priming and primed stimulation by OAG apparently invoke distinct transduction mechanisms other than protein kinase C translocation.

Altered oxidative product formation in neutrophils of patients recovering from therapy for acute leukemia.

During chemotherapy for acute leukemias, severe neutropenia allows acquisition of life-threatening infections that are difficult to clear with antibiotics alone. With return of myelopoiesis, even severe infections often improve dramatically. We have sequentially examined oxidative metabolic responses of polymorphonuclear leukocytes (PMNL) from 30 patients with acute leukemias before induction chemotherapy and after recovery of myelopoiesis (circulating PMNL greater than 500/microL). Maximal oxidative metabolic responses were quantitated by flow cytometric analysis of H2O2-dependent oxidation of intracellular 2',7'-dichlorofluorescin (DCFH) in individual PMNL after stimulation with phorbol myristate acetate (PMA). Resting PMNL oxidized a mean of 6.8 attomoles (amol) DCFH/cell/15 min, with no difference between normal or patients' PMNL. PMA-stimulated normal PMNL oxidized 183 +/- 35 amol/cell (mean +/- SD, n = 120). In patients' PMNL obtained before chemotherapy, the mean DCFH oxidation was not significantly different from controls (216 +/- 78 amol/cell). However, 11 of 22 samples revealed populations of granulocytes with increased (primed) oxidative responses; seven of these 11 patients had proven or suspected infection at presentation. At recovery from chemotherapy-induced neutropenia, PMNL from 19 of 21 patients possessed one or more significant subpopulations with primed oxidation in response to PMA. In these 19 patients, 61% +/- 8% of PMNL comprised primed populations that oxidized 503 +/- 46 amol/cell. Oxidative activity was most pronounced in patients with proven or clinically suspected infections (with 41% +/- 9% of PMNL oxidizing 615 +/- 79 amol/cell). However, oxidative responses to PMA were also significantly increased in recovery PMNL from ten patients without clinical or laboratory evidence of active infection (79% +/- 11% of PMNL primed to oxidize 402 +/- 29 amol/cell). The peak responses of the primed subpopulations were short-lived and generally lasted three days or less, although oxidative responses remained elevated above normal for a week or more. All of the patients with increased PMNL responsiveness survived their hospitalization. In contrast, PMNL from four patients had a significant population (18% to 82% of cells) with reduced responsiveness. Two of these four patients (with 71% and 75% subnormal cells) died during this induction attempt; the third died during a second induction attempt; only one survived to discharge. The clinical significance of these phenomena is yet to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)

Subpopulations of neutrophils with increased oxidative product formation in blood of patients with infection.

Stimulated human polymorphonuclear leukocytes (PMNL) have a marked increase in oxidative metabolism, producing reduced oxygen species (e.g., H2O2) that mediate bacterial killing. Previously, quantitation of metabolic responses of PMNL from patients with acute infections employed assays that measure mean activity of the entire PMNL population; such studies reported a modest and highly variable increase in oxidative metabolic responses of such "toxic" PMNL compared with normal cells. To assess metabolic capability of PMNL from 51 patients with acute bacterial infection, we employed a quantitative flow cytometric assay of H2O2-dependent oxidative product formation, the intracellular oxidation of 2',7'-dichlorofluorescin (DCFH). After stimulation by phorbol myristate acetate, the PMNL of patients demonstrated an increase in mean DCFH oxidation (315 +/- 14 and 180 +/- 4.5 amol/cell, patients and controls). Hexose monophosphate shunt activation was similarly increased in stimulated PMNL from bacteremic patients. These data are comparable with previous studies of mean metabolic activities of toxic PMNL. However, these mean values underestimate the quantitative responses of the hyperresponsive ("primed") PMNL within a mixture of normal and primed PMNL in the patients' blood. The flow cytometric assay demonstrated that the PMNL of the patients were composed of two populations. One population of PMNL had normal oxidative responses; the other "primed" population had up to 4.6 times the oxidative product formation of normal cells. Similar priming of circulating PMNL was caused by infection with gram-positive or gram-negative staining bacteria or by Candida species. The proportion and oxidative ability of the primed PMNL occurred independently of the number of juvenile neutrophil forms and independently of "toxic" morphologic changes of Wright's-stained PMNL. On the average, 40% of the PMNL of patients were primed, but the size of the primed PMNL population varied widely between patients (range 0 to 80%). This variable subpopulation may explain the variability of mean responsiveness of the PMNL of patients reported previously. Moreover, the marked increase in oxidative metabolic capability of the primed PMNL may be a significant component of the host response to acute infection. It could also contribute to the damage to host tissues such as pulmonary vascular endothelium during bacteremia.

Subinhibitory concentrations of antibiotics alter fibronectin binding to Staphylococcus aureus.

Fibronectin, a high-molecular-weight glycoprotein, is found in plasma and on mammalian cell surfaces. Recent reports have suggested that bacterial-fibronectin interactions play a role in bacterial attachment to host cells. Subinhibitory concentrations of lincosamines, erythromycin, and chloramphenicol decreased fibronectin binding to Staphylococcus aureus, whereas beta-lactam antibiotics enhanced this interaction.

Effects of subinhibitory concentrations of antibiotics on Staphylococcus aureus interactions with fibronectin.

Bacterial adherence to host tissues relies on interactions between tissue macromolecules and bacterial surface molecules. One of the major predisposing factors to infection with Staphylococcus aureus is trauma to tissues. A common element in traumatized tissues is fibronectin. In previous studies, we have shown that fibronectin binds to Staph. aureus. In this paper, we have investigated the effects of subinhibitory concentrations of antibiotics on fibronectin interactions with Staph. aureus. Exposure of Staph. aureus to 1/4 MIC of penicillin increases the number of binding sites and enhances adherence of Staph. aureus to a collagen-fibronectin matrix. Chloramphenicol, erythromycin, clindamycin, and U57,930E all decreased the number of binding sites. Also, U57,930E reduced Staph. aureus adherence to a collagen-fibronectin matrix. Taken together, these data suggest that penicillin may enhance Staph. aureus adherence to tissue fibronectin whereas U57,930E might reduce such binding.

Fibronectin binding to Staphylococcus aureus.

Bacteria are able to interact with a number of macromolecules which act as opsonins or tissue-adherence factors. Because soluble fibronectin may be important factors. Because soluble fibronectin may be important in the phagocytic removal of bacteria and insoluble fibronectin may serve as a bridge between bacteria and host tissues, we have characterized the binding of soluble plasma fibronectin to Staphylococcus aureus as a first step to understanding these interactions. The binding of 125I-fibronectin to clinical and laboratory strains of S. aureus was studied. Bound fibronectin was separated from free fibronectin by centrifugation. Specific binding was determined by subtracting the amount bound in the presence of excess fibronectin from the total amount bound. We found that (i) fibronectin bound saturably, irreversibly, and noncovalently to S. aureus when the binding reaction was carried out at pH 7.4 or greater; (ii) S. aureus harvested in logarithmic phase of growth from media buffered to pH 8.4, and from brain-heart infusion media which demonstrated the greatest number of fibronectin-binding sites; (iii) high molecular weight dextrans, fibrinogen, cyanogen bromide fragment 7 of collagen, cationic proteins, dibromide fragment 7 of collagen, cationic proteins, dithiothreitol, and protein A did not alt er fibronectin binding to S. aureus; (iv) nonsaturable binding occurred below pH 7.0 with peak binding occurring at pH 5.8; and (v) there were marked differences in the amounts of fibronectin that bind to different strains of S. aureus. S. aureus ATCC 25923, when harvested in logarithmic phase of growth from tryptic soy broth and tested for fibronectin binding in (2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline, pH 7.4, had 7500 binding sites/organism with an apparent association constant of 5.6 X 10(9) M-1.