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Biomedical subjects

L A Boxer

Publications and source records attributed to L A Boxer.

At least 91 records · Page 5Linked to original sources
PubMed1987

Deficiency of inducible suppressor cell activity in the Chediak-Higashi syndrome.

Peripheral blood lymphocytes from two Chediak-Higashi syndrome (CHS) patients were examined for their 1) natural killer (NK) cell functions 2) concanavalin A (Con A)-inducible suppressor cell activity, 3) soluble suppressor factor production, and 4) responsiveness to interferon alpha and interleukin-2 in comparison with age-matched normal controls. Peripheral blood lymphocytes or NK-enriched large granular lymphocytes from Chediak-Higashi syndrome patients showed negligible cytotoxic activity against several target cells. Although the NK activity of Chediak-Higashi syndrome lymphocytes could not be restored to normal levels by treatment with either interferon or interleukin-2, the percent enhancement of NK activity was higher for the patients than the controls. Soluble suppressor factor activity of culture supernates from the lymphocytes of Chediak-Higashi syndrome patients significantly inhibited the NK activity of allogeneic, normal peripheral blood lymphocytes, whereas lymphocytes from Chediak-Higashi syndrome patients precultured with Con A failed to suppress the cytotoxic activity of normal lymphocytes. These results demonstrate a previously unrecognized suppressor cell dysfunction in CHS patients.

Chediak-Higashi Syndrome↗
PubMed1987

Neutrophil C3bi receptors: formation of membrane clusters during cell triggering requires intracellular granules.

Video-intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescein- or rhodamine-labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double-labeling studies were conducted by first labeling with anti-CR3 followed by attachment to LPS/serum-treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum-treated slides. To determine the contribution of granule-associated CR3, we have studied neutrophils defective in receptor up-regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3-modified surfaces. The patient's neutrophils were defective in CR3 up-regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum-treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3-bearing granules at local sites of adhesion.

Blood Physiological Phenomena↗
PubMed1987

Neutrophil function disorders.

The polymorphonuclear leukocyte (neutrophil) is the most important phagocytic cell that defends the host against acute bacterial infection. Disorders of neutrophil function are suggested by recurrent cutaneous, periodontal, respiratory, or soft tissue infections. Staphylococcus aureus, gram-negative bacilli, and less commonly, Candida albicans, are the causative organisms. Treatment is supportive involving surgical drainage and antibiotics. Bone marrow transplantation offers hope to some patients. The biochemical and molecular defects have been identified for some of these disorders. Identification of these defects and their physiologic consequences have improved our understanding of how the activated neutrophil is attracted and adheres to inflammatory sites, and produces toxic products that destroy bacteria. However, the activated neutrophil may also damage normal tissue and participate in diseases such as rheumatoid arthritis and the adult respiratory distress syndrome (ARDS).

Bacterial Infections↗
PubMed1987

Relationship of the genes for Chediak-Higashi syndrome (beige) and the T-cell receptor gamma chain in mouse and man.

The genetic linkage of Chediak-Higashi syndrome and its murine analog, beige (bg), to the T-cell receptor (TCR-gamma) gamma chain gene is further defined. Previous studies using recombinant inbred strains of mice demonstrated that the murine bg gene is genetically linked to a murine TCR-gamma gene. We report that in the mouse the frequency of recombination between these two markers is 0.025. Further, we tested the hypothesis that these two genes are linked in the human genome by analyzing restriction fragment length polymorphisms (RFLPs) in five families with children afflicted with Chediak-Higashi syndrome. In three families, RFLPs in TCR-gamma genes were inherited discordantly from Chediak-Higashi syndrome, demonstrating nonlinkage. We postulate that there is an evolutionary chromosomal breakpoint between the bg gene and the TCR-gamma gene.

Animals↗
PubMed1987

In vitro and in vivo effects of treatment by platelet-activating factor on N-formyl-met-leu-phe-mediated responses of polymorphonuclear leucocytes.

Two chemoattractants, the peptide N-formyl-met-leu-phe (FMLP), and the ether phospholipid, platelet activating factor (PAF), each stimulate a variety of in vitro responses in polymorphonuclear leucocytes (PMN). Because often more than one inflammatory mediator is active during inflammation, we determined the effect on PMN of sequential stimulation with these two agents. Before FMLP stimulation, human PMN were exposed to PAF, at concentrations which gave little or no response when administered alone. PAF enhanced FMLP-elicited superoxide release in a dose-dependent fashion. Likewise, release of granular lysozyme from the cells was increased in PAF treated cells. Similar treatment with other phospholipids, including the lyso derivation of PAF, failed to produced these effects. Incubation with nordihydroguaiaretic acid, an inhibitor of arachidonic acid metabolism, had little effect on the enhancement of lysozyme release by PAF. To determine if enhancing effects by PAF might occur also in vivo, we studied rabbits receiving PAF and/or FMLP intravenously. When rabbits received 0.01 micrograms PAF (a dose which does not elicit the sustained neutropenia observed with higher doses of PAF) followed by 0.05 micrograms FMLP the absolute granulocyte count (AGC) dropped at 1 min (46 +/- 11% of original value), and continued to fall (24 +/- 12% at 10 min). Controls, treated with the suspending fluid for PAF, and then 0.05 micrograms FMLP, had a similar 1 min AGC value, but at 10 min AGC returned to 65 +/- 6.1% (P less than 0.001 for comparison of 10 min values). Thus PAF pretreatment enhanced FMLP-elicited granulocytopenia in vivo. Study of in vitro human PMN aggregation revealed that, at certain relative concentrations of PAF and FMLP, aggregation was enhanced. These studies show that both in vitro and in vivo responses of FMLP-stimulated PMN may be exaggerated by pre-exposure to PAF.

Animals↗
PubMed1987

Association between gelatinase release and increased plasma membrane expression of the Mo1 glycoprotein.

Mo1, a glycoprotein heterodimer (gp 155,95) that functions as an adhesion promoting molecule and as the C3bi receptor of human myeloid cells, is expressed in increased amounts in the plasma membrane after exposure of polymorphonuclear leukocytes (PMNs) to various stimuli. Previous studies have suggested that secondary granules represent an intracellular pool of Mo1 that, upon degranulation, fuse with the plasma membrane resulting in a tenfold increase in surface expression of Mo1. To determine the intracellular location of Mo1, we monitored Mo1 expression by immunofluorescence and compared it to the release of myeloperoxidase (MPO, a marker for the primary granules), vitamin B12 binding protein (B12BP, secondary granules), and gelatinase (gelatinase-containing organelles) following exposure to various stimuli. Human neutrophils stimulated with 20 mmol/L fluoride for 16 minutes exhibited a twofold increase in Mo1 expression and gelatinase release but no enhanced release of primary or secondary granular contents. In a similar fashion, incubation of cells at 37 degrees C for five minutes with 7.5 X 10(-9) to 10(-6) mol/L N-formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in significant increases in both surface Mo1 expression (three- to fivefold) and gelatinase release (five- to eightfold) without significant release of either MPO or B12BP. In addition, both the fluoride and FMLP experiments demonstrated that Mo1 up-modulation alone is not sufficient to activate superoxide (O2-) production. These data indicate that at least one intracellular storage pool of Mo1 is the gelatinase-containing organelles and that their fusion with the plasma membrane results in increased expression of Mo1 on the cell surface.

Fluorides↗
PubMed1987

Prevention of pulmonary injury in isolated perfused rat lungs by activated human neutrophils preincubated with anti-Mo1 monoclonal antibody.

Neutrophil activation results in neutrophil adherence and may subsequently cause lung injury through the generation of oxidants, release of granule proteases, and generation of a variety of mediator substances. We hypothesized that inhibition of neutrophil adherence and subsequent lung sequestration would attenuate the lung injury caused by activated neutrophils. Using isolated perfused rat lungs, we determined if anti-Mo1 monoclonal antibody (binds to the alpha subunit of a neutrophil glycoprotein [gp 155.94] that facilitates adherence) would attenuate lung neutrophil sequestration and lung injury caused by human neutrophils stimulated by phorbol myristate acetate (PMA). PMA-stimulated neutrophils but not PMA or neutrophils alone caused lung injury as assessed by accumulation of 125I-bovine serum albumin into lung parenchyma and alveolar lavage fluid. Incubation of neutrophils with anti-Mo1 antibody prior to stimulation with PMA attenuated lung injury and neutrophil sequestration. Furthermore, a histological survey revealed that anti-Mo1 antibody inhibited neutrophils present in the lung from spreading following exposure to PMA. Anti-Mo1 antibody did not inhibit PMA-stimulated neutrophil release of granule constituents or toxic O2 metabolites as evidenced by lysozyme and lactoferrin release or the reduction of ferricytochrome c in the lung perfusate. The inhibition of lung injury caused by the anti-Mo1 antibody was not likely due to a nonspecific effect of the antibody, since another murine monoclonal antibody of the same class (anti-Mo5) did not inhibit lung neutrophil sequestration or lung injury. Thus, in this experimental model, interference with the close approximation of the neutrophil to its target site inhibited the ability of the activated human neutrophil to cause injury.

Animals↗
PubMed1987

Measurement of antineutrophil antibodies by flow cytometry: simultaneous detection of antibodies against monocytes and lymphocytes.

We describe a flow cytometric technique which detects the presence of antineutrophil antibodies (NABs) in human serum. The technique provides a qualitative as well as a semiquantitative screen and provides an excellent method for monitoring the presence of antineutrophil antibodies in patients with suspected autoimmune neutropenia. Using light-scatter gating, individual populations consisting of neutrophils, monocytes, and lymphocytes can be examined simultaneously for the presence of antibody. The methodology utilizes an indirect immunofluorescence technique with FITC-labeled goat antihuman F(ab')2 antibody and fixed leukocyte suspensions. Furthermore, by utilizing class-specific FITC-labeled second antibody, significant information can be ascertained regarding the class, cell specificity, and quantity of detected antibody. Formalin fixation of neutrophils prevented pinocytosis of the fluorochrome, significantly reducing background fluorescence. Twenty-five normal subjects provided baseline antibody levels for each class. Of 92 patients with suspected autoimmune neutropenia, 27 had class IgG alone and seven were positive for both IgG and IgM class NABs. During treatment, IgG levels varied. IgM NABs alone were detected in four patients. Fifty-four patients had undetectable antibody. Antibodies were detected against monocytes in several of the IgG-positive patients. Two sera contained both IgA and IgG NABs. One serum contained IgA and IgG antibodies against monocytes. No IgD antibodies were detected in any sera tested. Some sera tested contained antibodies against lymphocytes--however, only in those sera which also contained antibodies to other cell types.

Autoantibodies↗
PubMed1987

Deficiency of leukocyte surface glycoproteins Mo1, LFA-1, and Leu M5 in a dog with recurrent bacterial infections: an animal model.

A dog with severe recurrent bacterial infections, impaired pus formation, delayed wound healing, and severe persistent leukocytosis was the result of a mother-son mating. Assessment of leukocyte function revealed profound abnormalities in adherence-dependent activities including impaired granulocyte adhesion to glass/plastic surfaces or nylon wool, decreased granulocyte aggregation and chemotaxis, and diminished lymphocyte blastogenesis, but normal neutrophil oxidative activity, serum immunoglobulin, and complement levels. By immunofluorescence analysis, CD11b and CD18 monoclonal antibodies specific for the 155-kd alpha polypeptide of Mo1 (gp 155, 94) and the 94 kd beta peptide common to Mo1, LFA-1 (gp 170, 94), and Leu M5 (p 150, 94) (surface molecules that promote leukocyte adhesion) failed to bind to unstimulated and A23187 calcium ionophore-stimulated granulocytes or mononuclear cells of the affected dog as compared with strong specific binding to canine control cells. The Mo1 glycoproteins were only barely detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of immunoprecipitates from lysates of 125I surface-labeled neutrophils from the affected dog as compared with intense bands seen with canine control cell precipitates. We conclude that this dog has a severe leukocyte surface glycoprotein deficiency syndrome that is similar, if not identical, to that recently recognized in humans. Dogs with deficiency of leukocyte Mo1, LFA-1, and Leu M5 expression may represent a useful animal model to characterize further the molecular basis for an inherited disorder in leukocyte effector function.

Animals↗
PubMed1987

Evidence that microenvironmental factors account for the age-related decline in neutrophil function.

We measured the function of neutrophils harvested from the supernatant of long-term marrow cultures in which stromal cell cultures derived from young mice were recharged with hematopoietic cells from old mice and vice versa. The functions measured were superoxide generation and enzyme secretion (lysozyme and glucuronidase), following cell activation by either phorbol myristate acetate (PMA) or Formyl-methionyl-leucyl-phenylalanine (FMLP). In addition we measured cytosolic calcium concentration and its increase following activation by FMLP. In all culture combinations recharge resulted in the recovery of greater than 2 X 10(6) cells/flask (95% neutrophils, 98% viable). Histologic studies of cytoplasmic markers indicated that recovered neutrophils were derived from the stem cell population employed for recharge. For each neutrophil parameter measured, function was markedly improved when old hematopoietic stem cells were recharged onto a young stroma and was significantly diminished when young stem cells were recharged onto an old stroma. This applied to superoxide generation, basal and stimulated enzyme levels, and to basal cytosolic calcium concentration and its increase following activation by FMLP. These results indicate that when old hematopoietic stem cells proliferate in a young microenvironment, neutrophil function returns virtually to normal. Conversely, function diminishes when young stem cells proliferate in an old stroma. These findings demonstrate, for the first time, that neutrophil function is modulated by microenvironmental factors, hormonal, cellular, or matrix, which are decreased in the elderly. That an age-related decline in function is extrinsic to the cell and is reversible has significance for the study of neutrophil function and of cellular aging and has potential therapeutic implications.

Aging↗
PubMed1986

Endogenous inhibitor of protein kinase C: association with human peripheral blood neutrophils but not with specific granule-deficient neutrophils or cytoplasts.

A Ca2+-activated and phospholipid-dependent protein kinase (PKC) has been described in several cell systems, including the human neutrophil. We found that less than 30 pmol 32P/10 min/10(6) cell equivalents of phorbol ester- or 1-oleoyl-2-acetylglycerol-stimulated PKC activity was obtained when neutrophil homogenates were used as an enzyme source. However, detergent-soluble and detergent-insoluble fractions prepared from the same homogenates, respectively, catalyzed 578 +/- 50 and 136 +/- 40 pmol 32P/10 min/10(6) cell equivalents. Recombining detergent-soluble and detergent-insoluble fractions resulted in the complete loss of activity. We therefore explored the possibility of an endogenous inhibitor of PKC in neutrophils. Homogenates from neutrophil cytoplasts, which lack the nuclei and intracellular granules of whole neutrophils, yielded 813 +/- 28 pmol 32P/10 min/10(6) cell equivalents. In addition, total neutrophil homogenates from a patient with a specific granule deficiency yielded high activities, namely 424 +/- 48 pmol 32P/10 min/10(6) cell equivalents. Specific granule-deficient neutrophils possessed translocated and activated PKC, and phosphoprotein patterns from these cells resembled those from activated normal neutrophils. Our results suggested the existence of an inhibitor of previously active PKC. That this inhibitor is primarily associated with neutrophil-specific granule membranes was suggested by our finding of high PKC activity associated with cell preparations or combinations of cell fractions that were free of specific granules, but not necessarily of other cellular organelles. Preliminary characterization of the endogenous inhibitor indicated that it was protease- and heat-sensitive, and did not exhibit either protease or phosphatase activity. We speculate that the inhibitor may play a physiologic role in regulating the activity of its target enzyme.

Cytoplasmic Granules↗
PubMed1986

Human neutrophils permeabilized with digitonin respond with lysosomal enzyme release when exposed to micromolar levels of free calcium.

We have recently reported that human neutrophils can be permeabilized with the cholesterol complexing agent saponin and that these cells can be induced to secrete the granule enzyme lysozyme in response to micromolar levels of free calcium. We now report that digitonin can be used in place of saponin and that it has several advantages. Permeabilization of human neutrophils was accomplished with 10 micrograms/ml digitonin in a high potassium medium. Normally impermeant solutes such as [14C]sucrose and inulin [14C]carboxylic acid gained access to one half of the intracellular water space marked with [3H]H2O. Between 30 and 100% of the cytoplasmic enzyme, lactate dehydrogenase, leaked from the intracellular space. The permeabilization process and calcium-triggered granule secretion were critically dependent upon temperature, time and digitonin concentration. Permeabilized neutrophils secreted beta-glucuronidase, lysozyme and vitamin B-12 binding-protein, constituents of both azurophil and specific granules, when exposed to micromolar levels of free calcium. Release of specific granule constituents appeared to be more sensitive to free calcium than release from azurophil granules. Although the amount of permeabilization varied considerably with each batch of cells, release of these granule markers was a consistent finding. Release of granule markers was accompanied by resealing of the cells to high-molecular-weight (Mr greater than 5000) solutes. Electron microscopic evidence also suggested that granule and plasma membranes were intact following digitonin treatment and that fusion of these membranes occurred in response to calcium. These results suggest that elevation of intracellular free-calcium levels is a sufficient condition for lysosomal enzyme release.

Calcium Chloride↗
PubMed1986

Oxidant activity in expired breath of patients with adult respiratory distress syndrome.

Hydrogen peroxide levels were measured in the breath condensate of 43 patients receiving mechanical ventilation. In 16 patients the mean breath condensate peroxide level was 1.68 +/- 0.35 mumol/l on the day they met diagnostic criteria for adult respiratory distress syndrome (ARDS). The peak breath condensate peroxide level in the 27 patients in whom ARDS did not develop was significantly lower (0.34 +/- 0.08 mumol/l). Plasma lysozyme, a measure of in-vivo neutrophil turnover, was significantly higher in ARDS than in non-ARDS patients (9.2 +/- 2.2 U/ml v 3.4 +/- 1.1 U/ml). These findings support the hypothesis that neutrophil activation and oxidant production are involved in the pathogenesis of ARDS.

Breath Tests↗
PubMed1986

Ultrastructural morphology and cytochemistry of iron-deficient polymorphonuclear leukocytes.

Previous studies have documented decreased activities of certain enzymes and altered function in polymorphonuclear leukocytes (PMN) during iron deficiency. The present study was undertaken to determine if the enzymatic abnormalities could be correlated with morphologic or quantitative change in PMN granules. Ultrastructural examination of primary and secondary granules and assessment of the secondary granule components alkaline phosphatase and vicinal glycol-containing glycoconjugates was performed in rabbit bone marrow, peripheral blood, and peritoneal heterophils. In addition, biochemical quantifications of the secondary granule component alkaline phosphatase and the primary granule marker beta-glucuronidase were performed. The results confirmed that a marked, significant decrease in alkaline phosphatase occurs in iron-deficient animals; however, no biochemical decrease in beta-glucuronidase activity was observed. Ultrastructurally, PMN secondary granules of iron-deficient rabbits tended to be more numerous than in controls when examined with morphometric and glycoconjugate staining methods, but lacked staining in alkaline phosphatase preparations. These results demonstrate that iron-deficient rabbits produce normal to increased quantities of primary and secondary granules, despite a uniform deficiency of alkaline phosphatase, a secondary granule marker.

Alkaline Phosphatase↗
PubMed1986

Neutropenia and thrombocytopenia: antibodies directed against circulating neutrophils and bone marrow myeloid progenitor cells (CFU-C).

The pathophysiology of a neutropenic disorder was evaluated in a 60-year-old man using myeloid progenitor cell (CFU-C), colony stimulating activity (CSA), and antineutrophil antibody assays. An immunoglobulin, present in the patient's serum at presentation, was directed against peripheral blood neutrophils, and autologous and allogeneic bone marrow CFU-C. A course of prednisone resulted in resolution of the neutropenia and a disappearance of the cytotoxic antibody. This study suggests that the patient's neutropenic disorder resulted from antibody-mediated destruction of circulating neutrophils and a suboptimal bone marrow granulopoietic response.

Agranulocytosis↗
PubMed1986

Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils.

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose-dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose-dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the "translocation" of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.

Calcium↗