J G Bender

Altered function of synovial fluid granulocytes in patients with acute inflammatory arthritis: evidence for activation of neutrophils and its mediation by a factor present in synovial fluid.

In rheumatoid arthritis (RA) a chronic inflammatory state exists in which the synovial fluid is periodically filled with large numbers of polymorphonuclear leukocytes (PMNs). Oxygen radicals produced by these cells have been implicated as mediators of tissue damage and may be directly involved in the pathogenesis of RA. We examined the production of oxygen radicals by synovial fluid PMNs (SFPMNs) and peripheral blood PMNs (PB-PMNs) by measuring chemiluminescence (CL) as well as Superoxide anion (O2−) release. Increased spontaneous CL in the presence of luminol and increased CL in response to phorbol myristate acetate (PMA) was observed in SF-PMNs when compared to PB-PMNs. When zymosan was used as the stimulus in the absence of luminol, a slightly lower CL response was observed in SF-PMNs as compared to PB-PMNs. No significant differences were observed in the generation of O2− generation with any stimulus. Preincubation of normal PBPMNs in 10% synovial fluid enhanced the luminol-dependent spontaneous and PMA-stimulated CL as well as zymosan-stimulated CL. When O2− release from normal PB-PMNs pretreated with 10% synovial fluid was compared to untreated controls, enhancement of spontaneous O2− release was observed. PMA- and zymosan-stimulated responses did not differ significantly from controls. Increased spontaneous and PMA-stimulated release of myeloperoxidase (MPO) was also observed in normal PB-PMNs pretreated with synovial fluid. These findings may explain the increased luminol-dependent CL since this type of CL requires the presence of MPO. Our findings suggest that the enhanced chemiluminescence observed in normal PMNs treated with synovial fluids may be related to increases in spontaneous O2− generation and myeloperoxidase release. Increased MPO release may account for enhanced CL observed in SF-PMNs.

Clinical impact of ex vivo differentiated myeloid precursors after high-dose chemotherapy and peripheral blood progenitor cell rescue

The infusion of ex vivo differentiated myeloid precursors may be able to shorten the period of obligatory neutropenia after high-dose chemotherapy and peripheral blood progenitor cell rescue by providing cells capable of differentiating to mature neutrophils within days of infusion. To test this hypothesis, 21 female patients with metastatic breast cancer underwent progenitor cell mobilization with cyclophosphamide, etoposide and G-CSF. CD34+ cells from one to two leukapheresis products were isolated and placed in suspension culture with a serum-free growth medium supplemented with PIXY321. The cultures were maintained for 12 days with subcultures initiated on day 7. The remaining leukapheresis products were cryopreserved in an unmanipulated state. Forty-eight hours after completing high-dose cyclophosphamide, thiotepa and carboplatin, the cryopreserved progenitors were infused, followed 1 to 24 h later by infusion of the differentiated myeloid precursors. In one patient, the cultured cells were labeled with Indium-111 with nuclear imaging performed up to 48 h post infusion. The differentiated myeloid precursors were suitable for infusion in 17 of the patients with a median 13-fold expansion of total nucleated cells. A range of 5.6 to 1066 × 107 nucleated cells were infused. Morphologically the cells were predominantly of myeloid lineage (63%) with a median 41% of the cells expressing CD15. No untoward effects were noted with the infusion of the cultured cells. The median days to neutrophil and platelet recovery were 8 and 10 days, respectively. There was a significant relationship (r = 0.67, P = 0.007) between the dose of differentiated myeloid precursors (CD15+ cells) and the depth and duration of neutropenia; a similar relationship, however, was also observed with the dose of cryopreserved CD34+ cells. After infusion of the radiolabeled myeloid precursors, a pattern of distribution similar to radio-labeled granulocytes was noted with uptake detected initially in the lungs and subsequently the reticulo-endothelial system. The impact of differentiated myeloid precursors on neutropenia as an adjunct to high-dose chemotherapy and peripheral blood progenitor cell rescue remains unclear from this study. Further study with controlled doses of cryopreserved progenitors and escalating doses of differentiated myeloid precursors is required. Bone Marrow Transplantation (2000) 26, 505–510.

A Model for the Regulation of Myelopoiesis by Specific Factors

Hematopoietic cells originate from stem cells that generate progenitor cells programmed to differentiate along specific cell lineages. Recently, a number of factors have been identified that are involved in regulating the proliferation of myeloid lineage cells. We propose a model in which a hierarchy of specific factors acts sequentially during defined stages of maturation to regulate myelopoiesis. The role of these factors as competence or progression factors in causing cells to enter and traverse the cell cycle is discussed. Experimental evidence supports the model.

Stimulus interactions in release of superoxide anion (O2-) from human neutrophils. Further evidence for multiple pathways of activation.

A wide variety of agents stimulate superoxide anion (O2−) release from human neutrophils. To determine whether the same or different cellular pathways are utilized, neutrophils were stimulated to release O2− with combinations of f-Met-LeuPhe (FMLP) (10−7 M), C5a (25 nM), Con A (100 μg/ml), arachidonic acid (100 μM), and PMA (1 μg/ml). These concentrations produced maximal O2− production when used alone. A synergistic response was observed when Con A was used in combination with FMLP or C5a. This response was twice the expected release in cytochalasin B-treated cells and three to five times the expected release in untreated cells. Additional studies showed that synergism was dependent upon the simultaneous presence of both agents. Additive O2− responses were observed when either FMLP, Con A, or C5a was tested in combination with arachidonic acid and when FMLP and C5a were tested together. When PMA was tested with C5a, FMLP, or Con A, a nonadditive O2− response resulted, whereas mixtures of PMA and arachidonic acid resulted in a less than additive response. These contrasting results using different soluble stimuli in combination suggest that multiple pathways exist for the stimulation of neutrophil O2− release, with some stimuli being totally independent of each other (possibly activating separate pools of oxidase), while other stimuli show cooperative effects on oxidase activation.

Relationship of chemotactic receptors for formyl peptide and C5a to CR1, CR3, and Fc receptors on human neutrophils.

The co‐expression of C5a and formyl‐methionine‐leucine‐phenylalanine‐lysine (FMLPL) receptors with CR1, CR3, and Fc receptors on human neutrophils (PMN) was studied. Fluorescein‐conjugated C5a (FL‐C5a) and FMLPL (FL‐FMLPL) were used to identify C5a and formyl peptide receptors. CR1, CR3, and Fc receptors were identified with monoclonal antibodies and a Texas red‐labeled goat anti‐mouse immunoglobulin second step reagent The co‐expression of chemotactic receptors with CR1, CR3, or Fc receptors was evaluated using two‐color flow cytometry. A direct correlation between the degree of expression of receptors for FL‐FMLPL and the expression of CR3, CR1, and Fc receptors on individual PMN was observed. In contrast, no correlation between the degree of C5a receptor expression and CR1, CR3, or Fc receptor expression was found. Similar results were obtained with PMN after up regulation of CR1, CR3, Fc, and FMLPL receptors by incubation at 37°C for 10 min with or without phorbol myristate acetate. These data suggest that the expression of FMLPL, CR1, CR3, and Fc receptors are regulated in a similar manner, whereas C5a receptor expression is regulated independently. Furthermore, these data indicate that within a given population of PMN, a parallel exists between the degree of CR1, CR3, FMLPL, and Fc receptor expression on individual cells.

Characterization of Human Mononuclear Cells Using Reduced Pyridine Nucleotide Fluorescence and Flow Cytometry

Peripheral blood monocytes undergo an oxidative burst similar to that seen in neutrophils. The basis for this response appears to be an NAD(P)H oxidase that utilizes reduced NAD(P)H to form superoxide anion. We utilized the unique UV‐stimulated fluorescence property of reduced pyridine nucleotides to analyze NAD(P)H utilization in monocytes. UV‐stimulated fluorescence in mononuclear cell preparations indicated two populations of cells with the highly fluorescent cells having a Coulter volume consistent with that of monocytes. Dual laser analysis with monoclonal antibodies confirmed that these highly fluorescent cells are monocytes by showing them to be OKM1 +, Leu DR +, and anti‐monocyte 0.2 +. Natural killer (NK) cells, as defined by Leu 7, were not found in this highly fluorescent population. Stimulation of mononuclear cells with phorbol myristate acetate caused a fluorescence loss indicative of NAD(P)H oxidation in monocytes but not in lymphocytes. Stimulation with suboptimal concentrations of PMA (1–5 ng/ml) resulted in a dose‐dependent fluorescence loss in monocytes that occurred in an all‐or‐none fashion identical to the pattern observed in neutrophils. Simultaneous measurement of H2O2 production using dichlorofluorescein formation with NAD(P)H fluorescence indicates that oxidant production occurs in a graded manner. This method, then, provides a convenient way to study in single cells the metabolic events involved in depletion and replenishment of NAD(P)H during the oxidative burst and demonstrates an additional means by which to distinguish monocytes from lymphocytes using flow cytometry.

Modulation of neutrophil-reduced pyridine nucleotide content following stimulation with phorbol myristate acetate and chemotactic factors.

Modulation of NAD(P)H in human neutrophils (PMN) following stimulation with phorbol myristate acetate (PMA) or chemotactic factors was determined by flow cytometry. Stimulation of PMN with 1 μg/ml of PMA results in a time‐dependent decrease in fluorescence, attributable to the oxidation of NAD(P)H. The decrease in fluorescence did not occur with PMN from a patient with chronic granulomatous disease (CGD) and was observed in only half of PMN from the mother of the patient. Loss of fluorescence in normal PMN was maximal following 7–15 min of stimulation with PMA. Simultaneous measurement of PMA‐stimulated NAD(P)H oxidation and H2O2 production showed that NAD(P)H oxidation occurred as an all‐or‐none response while H2O2 production showed a graded response.

Decreased opsonic activity forStaphylococcus aureus in neonatal and late gestation maternal sera

Heat-killedStaphylococcus aureus (ATCC strain 25923) andEscherichia coli K-12 were used as target microorganisms for opsonization by serum from neonates or mothers at various stages of pregnancy or postpartum. The level of opsonic activity was evaluated by titrating serum for the ability to coat bacteria for recognition by normal human PMN. Recognition of the organisms was quantitated by measuring the PMN Superoxide anion generation response to opsonized organisms. Studies show that opsonic activity forS. aureus was markedly decreased in serum from mothers in their second and third trimesters of pregnancy, in cord blood, and from infants at two weeks of age. Decreased activity was not observed in maternal serum in the first trimester of pregnancy and returned to control levels shortly after delivery. No significant difference was seen in opsonic activity forE. coli in these sera. The reduction in serum opsonic activity in mothers during the second and third trimesters of pregnancy suggests a systemic suppression of maternal immune responses during fetal development. This decreased activity is also observed in neonatal sera.