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L A Boxer

Publications and source records attributed to L A Boxer.

At least 37 records · Page 2Linked to original sources
PubMed1997

Severe chronic neutropenia: pathophysiology and therapy.

The development of recombinant-met human granulocyte-colony stimulating factor (r-metHuG-CSF) for clinical use has had a major influence on the treatment of many diseases. This impact has perhaps been greatest for treatment of severe chronic neutropenia (SCN) conditions for which there were no predictably effective treatment before the availability of these growth factors, particularly r-metHuG-CSF (Filgrastim, Amgen Inc, Thousand Oaks, CA; or Lenograstim, Rhone-Poulenc Rorer, Milan, Italy). Based on careful studies in many countries it is now known that more than 95% of these patients will respond promptly to r-metHuG-CSF treatment with normalization of the blood neutrophil levels and reduction in the occurrence of both major and minor consequences of their severe neutropenia. The availability of this treatment will undoubtedly lead to much additional research on the mechanisms governing neutrophil production and the basic mechanisms that can cause neutropenia among patients who have SCN. Among patients who have SCN those who are diagnosed to have severe congenital neutropenia (Kostmann's syndrome) or Shwachman-Diamond syndrome are at risk of developing myelodysplasia and/or acute myelogenous leukemia. The role of r-metHuG-CSF in facilitating the risk remains to be determined. Thus, it is important that long-term evaluation of the safety and efficacy of treatment of SCN and cooperation in research on these rare conditions proceed under the auspices of an international registry monitoring the clinical outcome of patients with severe congenital neutropenia.

Chronic Disease↗
PubMed1996

Tumor necrosis factor-alpha and FMLP receptors are functionally linked during FMLP-stimulated activation of adherent human neutrophils.

Human peripheral blood neutrophils (PMN) plated onto fibrinogen and activated with FMLP release H2O2 and lactoferrin, a specific granule component, with parallel kinetics. Although tumor necrosis factor-alpha (TNF alpha) only primes PMN in suspension, it is a potent agonist of adherent PMN. Activation of adherent PMN by FMLP (10(-7) mol/L) stimulated detectable release of TNF alpha within 45 minutes of stimulation, with maximal release (45.5 pg/10(6) cells) detected by 90 minutes. TNF alpha release paralleled the release of both lactoferrin and H2O2. To determine if TNF alpha plays a role in H2O2 and lactoferrin release, we investigated the effect of anti-TNF alpha antibodies on FMLP-stimulated activation of adherent PMN. A neutralizing rabbit anti-TNF alpha antibody inhibited both H2O2 and lactoferrin release stimulated by FMLP, whereas rabbit lgG, anti-HLA-A,B,C, anti-CD 14, and anti-interleukin-8 antibodies were without effect. The simultaneous addition of TNF alpha (1,000 U/mL) with anti-TNF alpha antibody reversed the inhibition seen with anti-TNF alpha alone. Furthermore, treatment of PMN with either actinomycin D or cylcoheximide resulted in partial (33%) inhibition of H2O2 and lactoferrin release, suggesting that protein synthesis is required for FMLP-mediated activation of adherent PMN. The addition of TNF alpha to either cycloheximide or of actinomycin D-treated PMN overcame the inhibition, indicating that the effect was specific for TNF alpha. The addition of antibodies against either the 55-or 75-kD TNF alpha receptors (referred to as p55 and p75, respectively) resulted in partial (32%) inhibition of FMLP-mediated activation of H2O2 and lactoferrin release, whereas a combination of both antibodies reduced their release to control levels. These data indicate that both p55 and p75 are involved in FMLP activation of adherent PMN. Taken together, these findings indicate that the production of TNF alpha and ligation of TNF alpha receptors are central to FMLP activation of PMN adherent to fibrinogen.

Animals↗
PubMed1996

Transforming growth factor-beta1 stimulates degranulation and oxidant release by adherent human neutrophils.

The signal transduction pathways that are activated by cytokines and growth factors binding to their receptors on human neutrophils (PMN) are poorly understood. When PMN in suspension encounter many of these agonists they are not activated, but rather are primed for subsequent activation. We and others reported that when PMN are plated onto fibrinogen and stimulated with cytokines or with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) they respond by releasing hydrogen peroxide (H202) and the specific granule component lactoferrin. Transforming growth factor-beta1 (TGF-beta1) is released by many cells including PMN. It has been reported that TGF-beta1 stimulates chemotaxis but not exocytosis or superoxide production by cells in suspension. We hypothesized that TGF-beta1 would activate PMN to release H202 when they were adherent to fibrinogen, a response mediated by beta2++integrin receptors. In this study, we determined whether TGF-beta1 stimulated H202 and lactoferrin release by PMN adherent to fibrinogen. TGF-beta1 stimulated H202 and lactoferrin release from adherent PMN in a concentration-dependent manner, with effects seen in the range of 0.1 to 100 pg/mL. Both H202 and lactoferrin release were detected by 60 min and continued for at least 180 min. Adhesion and spreading of PMN paralleled H202 and lactoferrin release. Ethanol (200 mM) blocked both H202 and lactoferrin release, suggesting the involvement of the phospholipase D pathway. In PMN labeled with lyso-[3H]phosphatidylcholine, we observed that TGF-beta1 treatment caused an increase in [3H]phosphatidate. Propranolol (150 microM), an inhibitor of phosphatidate phosphohydrolase, blocked both H202 and lactoferrin release, suggesting that the conversion of phosphatidic acid to diradylglycerol is an important step in PMN activation by TGF-beta1. Overall, these results are similar to those reported for fMLP activation of adherent PMN and suggest that a common pathway is involved in both chemoattractant and cytokine activation.

Butanols↗
PubMed1995

Human urokinase-type plasminogen activator primes neutrophils for superoxide anion release. Possible roles of complement receptor type 3 and calcium.

Urokinase-type plasminogen activator (uPA), which binds to cells via a specific receptor (uPAR), participates in pericellular proteolysis during leukocyte migration. Previous studies have indicated that uPAR is physically associated with CR3 (CD11b/CD18). To test the functional interactions of CR3 and uPAR, we have examined the ability of uPA to elicit changes in cytosolic calcium levels of normal neutrophils, neutrophils from a leukocyte adhesion deficiency (LAD) patient, and 3T3 transfectants expressing CR3, uPAR, or both. We found that calcium levels of neutrophils increased from 106 +/- 6 nM in untreated cells to 199 +/- 25 nM in the presence of uPA. In contrast, no significant change in calcium was observed when neutrophils from an leukocyte adhesion deficiency patient were examined. The uPA-dependent calcium rise was inhibited by mAb directed against either CR3 or uPAR and required intact uPA. To substantiate further these findings, we prepared transfectants expressing genes encoding uPAR, CR3, and both receptors; only cells expressing both receptors experienced a rise in intracellular calcium. Although uPA's calcium signal is insufficient to trigger superoxide production, FMLP dose-dependent superoxide production was greatly enhanced by incubating neutrophils with intact, but not fragmented, uPA. Flow cytometry experiments utilizing an FMLP analogue exclude the possibilities that urokinase binds to the FMLP receptor or up-regulates its expression. We suggest that calcium is a second messenger of uPA, that this message is mediated in a CR3-dependent fashion, and that this signal primes neutrophils for superoxide production.

3T3 Cells↗
PubMed1994

Contrasting effects of recombinant human granulocyte-macrophage colony-stimulating factor (CSF) and granulocyte CSF treatment on the cycling of blood elements in childhood-onset cyclic neutropenia.

Recombinant human granulocyte colony-stimulating factor (G-CSF) treatment has been shown to increase average neutrophil counts substantially in patients with childhood-onset cyclic neutropenia (or "cyclic hematopoiesis"), but not to eliminate the cyclic oscillations of neutrophil counts or those of other blood elements (monocytes, platelets, eosinophils, and reticulocytes) that are characteristic of this hematopoietic disorder. Indeed, oscillations of neutrophil counts are amplified during G-CSF treatment. We have compared the effects of recombinant granulocyte-macrophage-CSF (GM-CSF) with those of G-CSF in three patients with this disease (2 men and 1 woman, 17, 30, and 32 years of age). These patients were treated with GM-CSF (2.1 micrograms/kg/day, subcutaneously) for 6 weeks, preceded and followed by 6 to 13 weeks of detailed observation to document changes in the cyclic oscillations of blood neutrophils and other blood elements; two of the patients were subsequently treated with G-CSF (5.0 micrograms/kg/d, subcutaneously) and observed for comparable periods of time. Unlike G-CSF treatment, which increased average neutrophil counts more than 20-fold, GM-CSF increased neutrophil counts only modestly, from 1.6- to 3.9-fold, although eosinophilia of varying prominence was induced in each patient. However, at the same time, GM-CSF treatment dampened or eliminated the multilineage oscillations of circulating blood elements (neutrophils, monocytes, platelets, and/or reticulocytes) in each of the patients. In contrast, G-CSF treatment of the same patients markedly amplified the oscillations of neutrophil counts and caused the cycling of other blood elements (monocytes in particular) to become more distinct. These findings support the conclusion that the distinctive cycling of blood cell production in childhood-onset cyclic neutropenia results from abnormalities in the coordinate regulation of both GM-CSF-responsive, multipotential progenitor cells and G-CSF-responsive, lineage-restricted, neutrophil progenitors.

Adolescent↗
PubMed1994

Ceramide regulates oxidant release in adherent human neutrophils.

We investigated the role of sphingolipids in regulating oxidant release in adherent human neutrophils. Stimulation of adherent neutrophils with formyl-Met-Leu-Phe (fMLP) resulted in the accumulation of ceramide at a time when H2O2 release is terminated. H2O2 release in fMLP-stimulated neutrophils was suppressed in a concentration-dependent manner by the exogenous addition of several free sphingoid amines and short chain ceramides. Sphingosine, dihydrosphingosine, phytosphingosine, N-acetylsphingosine, and N-acetylphytosphingosine, but not N-acetyldihydrosphingosine, inhibited formyl peptide-stimulated oxidant release. The half-maximal inhibitory concentrations of N-acetylsphingosine and N-acetylphytosphingosine were 0.51 and 0.38 microM, respectively. Sphingosine, dihydrosphingosine, and phytosphingosine were less potent inhibitors with half-maximal inhibitory concentrations of 1.78, 15.4, and 1.48 microM, respectively. The 4 beta-phorbol 12 beta-myristate 13 alpha-acetate-induced respiratory burst was inhibited by 5 microM of sphingosine but not by 5 microM of N-acetylsphingosine. The effects of N-acetyl-conjugated sphingols (C2 ceramides) on phosphatidylcholine-specific phospholipase D and phosphatidic acid phosphohydrolase were markedly different from the effects of the related sphingoid bases. Both C2 ceramides and sphingoid bases partially inhibited the diradylglycerol formation by the phosphatidylcholine-specific phospholipase D pathway. Under the same conditions, however, N-acetyldihydrosphingosine and dihydrosphingosine failed to suppress H2O2 release in fMLP-stimulated neutrophils. These findings demonstrate that C2 ceramides inhibit H2O2 generation in fMLP-stimulated neutrophils via protein kinase C- or sphingoid base-independent mechanisms. The effect of ceramide in inhibiting the respiratory burst is structurally specific, because either a 4,5-trans double bond or 4-hydroxyl group is required for the inhibition. Therefore, ceramides may regulate oxidant release in adherent neutrophils.

Cell Adhesion↗
PubMed1994

Phospholipase D-mediated diradylglycerol formation coincides with H2O2 and lactoferrin release in adherent human neutrophils.

Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the onset of the respiratory burst in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP). Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin-containing specific granules. Since diradylglycerol (DRG) has been implicated in PMN secretion and oxidant release, we measured DRG formation during PMN adhesion to fibrinogen. PMNs were added to fibrinogen-coated plastic in the presence of fMLP, and H2O2 release, lactoferrin release, and DRG formation measured over a time course of 120 min. H2O2 and lactoferrin release were not apparent until 45-60 min, reaching maximal levels by 120 min. In contrast, DRG concentration increased by 15-30 min, from 275 +/- 27 pmol/mg of protein in resting cells to 600 +/- 173 pmol/mg protein in cells exposed to fMLP. DRG levels returned to base line by 30-45 min (383 +/- 32 pmol/mg of protein) before increasing again between 60 and 120 min (944 +/- 230 pmol/mg of protein and 1632 +/- 351 pmol/mg of protein, respectively). Propranolol, an inhibitor of phosphatidate phosphohydrolase, caused a dose-dependent inhibition of both H2O2 and lactoferrin release, with maximal inhibition at 50-100 microM. Propranolol also inhibited the second, but not the first phase of DRG formation. Similarly, ethanol treatment completely blocked H2O2 and lactoferrin release, and the second phase of DRG formation. In the presence of ethanol, phospholipase D (PLD)-mediated formation of [3H]phosphatidylethanol from 3H-O-alkyl-phosphatidylcholine corresponded to the second, but not the first, phase of DRG formation (23,169 +/- 2,017 cpm/mg protein, ethanol versus 2,696 +/- 261 cpm/mg protein, control). These data indicate that DRG, generated through the activation of PLD, plays an important role in degranulation and oxidant release in adherent PMNs.

Cell Adhesion↗
PubMed1994

Granulocyte colony-stimulating factor (G-CSF) production and G-CSF receptor structure in patients with congenital neutropenia.

Congenital neutropenia (Kostmann's syndrome [KS]) is an autosomal recessive syndrome that is characterized by profound neutropenia, resulting in major clinical infections and death. Since the neutropenia and symptoms in KS improve in response to exogenous administration of granulocyte colony-stimulating factor (G-CSF), we studied bone marrow cytokine (G-CSF, granulocyte-macrophage CSF [GM-CSF], and interleukin-6) production under both basal and stimulated conditions. No differences in G-CSF, GM-CSF, or IL-6 gene expression were found in bone marrow stromal cells between normal controls and KS patients, and all three cytokines were detected by enzyme-linked immunosorbent assay (ELISA) in medium conditioned by bone marrow stromal cells from normal donors and patients with KS. Each KS patient tested had detectable, functional G-CSF in their own serum before exogenous G-CSF administration. Since G-CSF production appeared normal in KS patients, we then asked whether we could detect structural defects in the signaling portion of G-CSF receptor genes. Polymerase chain reaction (PCR) amplification of the G-CSF receptor transmembrane region alone, and of the transmembrane plus cytosolic portions of the receptor, yielded the size products predicted from the sequences of the normal G-CSF receptor. Single-strand conformational polymorphism (SSCP) analysis of G-CSF receptor PCR products demonstrated no variance in structural conformation between KS patients and normal subjects. These results demonstrate that bone marrow stromal cells in patients with KS secrete normal concentrations of functional G-CSF and suggest that the neutropenia in KS patients is caused by an inability of neutrophilic progenitor and precursor cells to respond to normal, physiologic levels of G-CSF. Such a defect, clinically responsive to pharmacologic doses of G-CSF, might be caused by defects in the post-G-CSF receptor signal transduction pathway.

Base Sequence↗
PubMed1994

Aberrant capping of membrane proteins on neutrophils from patients with leukocyte adhesion deficiency.

Several functional defects have been found in neutrophils from leukocyte adhesion deficiency (LAD) patients who fail to express the CD11/CD18 leukoadhesins: Mo1, LFA-1, and p150,95. To better understand the functional defects of LAD neutrophils, we have performed capping experiments. Purified normal or LAD neutrophils were labeled with fluorochrome-conjugated concanavalin A (Con A) or F(ab')2 fragments of antiurokinase-type plasminogen activator receptor (uPAR), anti-Fc gamma RIII (CD16), anti-Mo5, and anti-CD14 antibodies. F(ab')2-labeled cells were capped using a second-step F(ab')2 fragment of an antimurine Fab antiserum. Cells were capped for 30 minutes at 37 degrees C, then observed by fluorescence microscopy. LAD neutrophils were found to be deficient in capping, but not clustering of all of the reagents tested to date. The percent of cells exhibiting capping of Con A, Fc gamma RIII, urokinase receptor, CD14, and Mo5 were 52%, 67%, 70%, 25%, and 64% for normal neutrophils but were only 10%, 5%, 2%, 3%, and 1%, respectively, for LAD neutrophils. Capping of this panel of membrane components in LAD or normal neutrophils was not augmented by the addition of either 10(-5) mol/L colchicine or 10(-7) mol/L FMLP. Because capping requires membrane-to-cytosol communication and an intact microfilament linkage, we suggest that leukoadhesins may play a broad role in promoting the redistribution of membrane components including adherence-related receptors such as Fc gamma RIII and the urokinase receptor.

Animals↗
PubMed1994

Exocytosis of a subpopulation of specific granules coincides with H2O2 production in adherent human neutrophils.

Unlike polymorphonuclear leukocytes (PMN) that are activated in suspension, PMN plated onto extracellular matrix (ECM) proteins exhibited a prolonged delay in the onset of the respiratory burst in response to PMA, FMLP, A23187, or GM-CSF. The present study was focused on examining the events leading to H2O2 release in adherent PMN. The time course of stimulated H2O2 release from PMN plated on fibrinogen (FG) kinetically paralleled cell spreading and lactoferrin release. In contrast, the release of another specific granule component, vitamin B12-binding protein, preceded H2O2 generation by at least 20 min, suggesting the differential mobilization of subpopulations of specific granules. FMLP-stimulated PMN from a patient with leukocyte adhesion deficiency (LAD), lacking CD11/CD18 integrins and unable to adhere to FG, attached and spread on thrombospondin (TSP) and generated substantial amounts of H2O2. However, PMN from a second LAD patient, able to attach but not spread on TSP, failed to generate oxidant. These data indicated that PMN spreading might be a prerequisite for H2O2 generation and that CD11/CD18 integrins are likely not the only surface receptors involved in this response. Exposing PMN to the intracellular Ca2+ chelators MAPTAM or BAPTA significantly reduced H2O2 generation in response to the receptor-mediated agonists, FMLP or GM-CSF, but did not affect PMA-stimulated H2O2 generation. Under conditions that resulted in reduced H2O2 generation (i.e., MAPTAM or BAPTA plus FMLP or GM-CSF), lactoferrin, but not vitamin B12-binding protein, release was also reduced, suggesting a link between the secretion of lactoferrin-containing specific granules and H2O2 production. Since neither MAPTAM or BAPTA blocked PMN spreading, it appeared that cell spreading alone was not sufficient for H2O2 production. Thus, the major requirement for H2O2 generation appeared to be the exocytosis of a distinct population of lactoferrin-containing specific granules. In support of this observation, PMN-like HL-60 cells, which lack specific granules, attached and spread on FG but failed to release H2O2. Thus, we postulate that the delivery of cytochrome b from lactoferrin-containing specific granules to the plasma membrane during activation contributes to oxidant production in PMN adherent to ECM proteins.

Cell Adhesion↗
PubMed1994

Role of diradylglycerol formation in H2O2 and lactoferrin release in adherent human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the release of H2O2 in response to fMLP. Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin-containing specific granules and activation of phospholipase D (PLD). We also found that chelation of intracellular calcium blocked both lactoferrin and H2O2 release in stimulated PMNs in spite of the fact that adhesion and spreading remained normal. Since diradylglycerol (DRG) formation has been implicated in PMN secretion and oxidant release, we determined the effect of intracellular calcium chelation on PLD activation and DRG formation to ascertain whether DRG formation was coupled to lactoferrin and H2O2 release. We observed that chelation of intracellular calcium with bis-(O-aminophenoxy)-ethanol-N,N;N'-tetraacetic acid (BAPTA) prevented PLD activation as monitored by inhibition of phosphatidylethanol formation. Formation of DRG derived from phosphatidic acid (PA) was also inhibited in the presence of BAPTA. Following the addition of the calcium ionophore ionomycin to the BAPTA-treated PMNs, lactoferrin and H2O2 release was coincident with the onset of DRG formation. Also the addition of sn-1,2-didecanoylglycerol to the BAPTA-treated PMNs stimulated them to release H2O2. Our studies support the hypothesis that DRG derived from PLD activation is required for degranulation of specific granules and associated H2O2 release from adherent PMNs.

Amino Acid Sequence↗
PubMed1993

Regulation of interleukin 8 gene expression by oxidant stress.

Interleukin 8 (IL-8) is a recently described cytokine that functions as a potent neutrophil chemoattractant and activator. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) and the regulation of IL-8 gene expression to specifically test the hypothesis that ROI would induce production of IL-8 mRNA and protein. In lipopolysaccharide-stimulated human whole blood, the OH radical scavenger dimethyl sulfoxide (Me2SO) dramatically inhibited (approximately 90%) IL-8 production, but had minimal effects on the production of tumor necrosis factor, interleukin 1 beta (IL-1), and IL-6. To determine whether NADPH-oxidase-generated free radicals were critical in the regulation of IL-8, studies were performed using blood from patients with chronic granulomatous disease. In both normal individuals and patients with chronic granulomatous disease, production of IL-8 could be initiated with lipopolysaccharide, phytohemagglutinin, or aggregated immune complexes, and this production could be inhibited by Me2SO (1% v/v). To examine if oxidant stress represents a ubiquitous mechanism for the induction of IL-8, experiments were performed in cultured cell lines. In the human hepatoma cell line Hep-G2, Me2SO dose-dependently inhibited tumor necrosis factor-stimulated IL-8 production, with a 74 +/- 1% reduction observed at a Me2SO concentration of 1%. Direct exposure to ROI demonstrated that H2O2 stimulated IL-8 production in a dose-dependent manner in Hep-G2 cells, A549 pulmonary type II epithelial cells, and human skin fibroblasts; this induction could be prevented by addition of catalase. The production of IL-8 appeared to be specific to an oxidant stress since exposure of the cells to heat shock or chemical stress did not induce expression of IL-8. These studies demonstrate that oxidant stress is an important regulator of IL-8 gene expression and support the hypothesis that low levels of ROI may serve to initiate IL-8 production which then serves to recruit neutrophils to sites of inflammation.

Antioxidants↗
PubMed1993

Lectin-like inhibition of immune complex receptor-mediated stimulation of neutrophils. Effects on cytosolic calcium release and superoxide production.

We have tested the role of lectin-like interactions, with particular emphasis on CR3, in insoluble immune complex (IC)-mediated activation of human polymorphonuclear leukocytes (PMN). The ability of IC to trigger intracellular Ca2+ release and O2- production by normal PMN, saccharide-treated cells, and CR3-deficient PMN (leukocyte adhesion deficiency, LAD, patients) were tested. When indo-1-labeled normal PMN were stimulated with IC in Ca(2+)-free buffer, intracellular Ca2+ rose from approximately 100 nM to approximately 230 nM. However, when LAD PMN were tested, a small rise in intracellular Ca2+ was observed. Because previous studies have shown that certain saccharides inhibit CR3-Fc gamma RIII co-capping, we tested a panel of saccharides to determine their ability to influence IC-mediated intracellular Ca2+ release. When normal PMN were exposed to 0.15 M N-acetyl-D-glucosamine (NADG), D-mannose, or alpha-methyl-mannoside, the Ca2+ response to IC was significantly reduced. However, addition of 0.15 M glucose, raffinose, sucrose, galactose, fructose, or sorbitol did not significantly affect the Ca2+ response, suggesting that the response was specific for certain saccharides. To test the physiologic consequences of these Ca2+ signals, we have examined the ability of saccharides to influence O2- production by normal PMN and the ability of LAD PMN to produce O2- upon triggering by IC. Normal PMN stimulated with IC generated 4.3 +/- 1.7 nmol/10(6) cells/30 min of O2-. In contrast, O2- production was inhibited by 0 to 20% by glucose, galactose, sucrose, sorbitol, fructose, and raffinose and > or = 50% by NADG and mannose. LAD PMN, which display diminished Ca2+ signals, were found to produce O2- at 47 +/- 6% of control levels. NADG and mannose dose-response studies indicated that they cooperatively block O2-.

Adult↗
PubMed1993

Simultaneous calcium-dependent delivery of neutrophil lactoferrin and reactive oxygen metabolites to erythrocyte targets: evidence supporting granule-dependent triggering of superoxide deposition.

Optical microscopic techniques have been utilized to study the deposition of lactoferrin, a specific granule marker, and superoxide anions into target erythrocytes during antibody-dependent phagocytosis. Previous studies from this laboratory have shown that the entry of superoxide anions into erythrocytes can be sensitively monitored with Soret band transmitted light microscopy. When neutrophils were incubated with BAPTA/AM, an intracellular Ca2+ chelator, they phagocytosed IgG-opsonized sheep red blood cells (SRBC) but did not affect the microscopically detected absorption of their Soret band. When these same erythrocytes were observed after the infusion of 20 microM ionomycin, a Ca2+ ionophore, 58% of the cell-bound SRBC targets were destroyed immediately. However, neutrophils from chronic granulomatous disease (CGD) patients were unable to affect the Soret absorption of erythrocyte targets under any conditions. These results suggest that a Ca2+ signal can participate in triggering superoxide deposition in targets. Since Ca2+ signals are known to participate in the exocytic release of granules, we tested the hypothesis that specific lactoferrin-bearing granules are delivered to targets in parallel with superoxide anions. Lactoferrin delivery to phagosomes was monitored using resonance energy transfer (r.e.t.) microscopy. SRBCs were opsonized with both unconjugated and rhodamine B isothiocyanate (RBITC)-conjugated rabbit anti-SRBC IgG. After incubation with adherent neutrophils, the samples were washed, fixed with 3.7% paraformaldehyde, then labeled with fluorescein isothiocyanate (FITC)-conjugated antilactoferrin IgG. Energy transfer between FITC and RBITC was imaged microscopically and quantitated by photon counting. Significant levels of r.e.t. between antilactoferrin and anti-SRBC labels were observed after phagocytosis, but not in the absence of acceptor fluorochromes. To control for r.e.t. specificity, neutrophil membranes were labeled with FITC-conjugated, anti-HLA IgG after internalization of rhodamine B-tagged SRBCs (RSRBCs). Although r.e.t. between lactoferrin and RSRBCs labels was observed, no r.e.t. between HLA and RSRBC labels could be found. Further studies showed that treatment of neutrophils with BAPTA inhibited r.e.t. between anti-lactoferrin and RSRBCs. However, addition of ionomycin relieved this inhibition of energy transfer. These experiments show that both lactoferrin and superoxide delivery to targets are regulated in parallel by a Ca(2+)-dependent pathway. Furthermore, by combining Soret microscopy with r.e.t. microscopy, we have shown that superoxide anions and lactoferrin are delivered to the same phagosomes. We speculate that the NADPH oxidase, which produces superoxide anions, is assembled on specific granule membranes, thus accounting for their parallel Ca(2+)-dependence, activation, and delivery.

Calcium↗
PubMed1993

Deposition of reactive oxygen metabolites onto and within living tumor cells during neutrophil-mediated antibody-dependent cellular cytotoxicity.

In this study we test the hypothesis that reactive oxygen metabolites are delivered from neutrophils to simultaneously both the cell surface and cytosol of opsonized YAC erythroleukemic target cells. Using 5' (or 6') carboxyl-2',7'-dichlorodihydrofluorescein (H2-CDCF) diacetate as starting material, we synthesized its succinimidyl ester derivative. H2-CDCF-conjugated IgG prepared from the succinimidyl ester derivative was used to opsonize targets. In vitro studies have shown that H2-CDCF becomes fluorescent upon exposure to reactive oxygen metabolites, including hydrogen peroxide. Using video intensified epifluorescence microscopy, we observed that reactive oxygen metabolites are deposited on tumor cell membranes during neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC). This deposition process is catalase sensitive. The role of reactive oxygen metabolites produced by neutrophils in triggering the oxidation of H2-CDCF is further supported by the observation that neutrophils from chronic granulomatous disease (CGD) patients did not affect target fluorescence. YAC tumor cells were also labeled with dihydrorhodamine 123 or dihydrotetramethylrosamine. The oxidized forms of these reagents were found within the cytoplasm of YAC cells. During ADCC normal neutrophils, but not neutrophils obtained from CGD patients, triggered the oxidation of dihydrorhodamine 123 and dihydrotetramethylrosamine within tumor cells. Using two-color automated epifluorescence microscopy, we could not detect temporal intermediates with fluorescence in only one compartment, i.e., either solely on the plasma membrane or in the cytoplasm. These observations suggest that reactive oxygen metabolites cross target membranes (< 12 sec. These studies show that reactive oxygen metabolites are deposited both onto and into tumor cells during ADCC, wherein both compartments could become vulnerable to oxidant-mediated damage.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1993

Volume of the spleen in children as measured on CT scans: normal standards as a function of body weight.

OBJECTIVE: The purpose of this study was to develop standards for the normal volume of the spleen in children as measured on CT scans. SUBJECTS AND METHODS: CT scans were used to measure the volume of the spleen in 48 children (30 boys and 18 girls), 1 day to 18 years old (mean and median ages were 8.2 and 7.8 years, respectively). Children who had underlying malignant tumors, infection, hematologic diseases, or other conditions that could alter splenic size were excluded. The area of the spleen on each axial section was determined by tracing its outline on the CT monitor and measuring the area of the region of interest. The area of the spleen on each section was multiplied by the slice thickness to calculate the volume of the spleen for each section. The total volume of the spleen was then determined by adding the individual volumes for each of the sections through the spleen. This method of calculating splenic volume has been validated in previous studies in adult subjects. The volume of the spleen was analyzed as a function of both body weight and age. RESULTS: The volume of the spleen correlated better with body weight than with age. The best regression model (r = .85) was a linear relationship as follows: splenic volume (cm3) = 0.7 + [4.6 x weight (kg)]. Using these data and a regression model, we generated standards for normal volume of the spleen as a function of body weight (with 95% tolerance intervals). CONCLUSION: We have developed CT standards for normal splenic volume in children. These standards can be used to objectively measure the size of the spleen in children who have clinically suspected splenomegaly.

Body Weight↗