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D A Stephany

Publications and source records attributed to D A Stephany.

15 recordsLinked to original sources
PubMed1996

Identification of surface molecules associated with physiologic activation of eosinophils. Application of whole-blood flow cytometry to eosinophils.

Activation is central to the eosinophil's functional role as an immune responder cell. To evaluate such activation in cells freshly isolated from peripheral blood, a method for whole-blood immunostaining and flow cytometry-based eosinophil selection was developed. Simultaneous comparison of purified eosinophils and whole-blood cells revealed significant differences in the levels of expression of various surface molecules, which suggested that the purification process activated the eosinophils. Subsequent analyses were conducted with the whole-blood assay. When eosinophils from helminth-infected persons (n = 18) were compared with those from normal individuals (n = 10), the early activation marker CD69 was found to be significantly increased (geometric mean (GM) = 4.3 vs. 1.0%, p = 0.04). The granulocyte activation marker CD66 was also up-regulated on eosinphils from helminth patients (GM = 53.3 vs. 31.0%, p = 0.044), as was the tetraspan family molecule CD81 (TAPA-1; GM = 79.4 vs. 48.2%, p = 0.02). Conversely, in vivo CD23 (FcepsilonRII) expression on eosinophils was decreased in the presence of parasitic infection (GM = 0.9 vs. 5.7%, p = 0.02). Expression of the eosinophil surface molecules CD69, CD81, and CD23 was significantly enhanced after cytokine stimulation in vitro with IL-3 or GM-CSF. In vivo, specific anthelmintic therapy resulted in decreased CD66 and CD25 expression (p < 0.05 compared with pretreatment) to levels approaching those seen in uninfected normal individuals. These findings indicate the dynamic nature of eosinophil surface molecules and demonstrate an important role for whole-blood staining in developing an understanding of the nature of eosinophil activation and of their role in inflammatory reactions to helminth parasites.

Anthelmintics↗
PubMed1989

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. IV. Immune reconstitution.

Studies of postmyeloablative immune reconstitution have been reported for allogeneic bone marrow transplantation and also for non-T cell-depleted autologous/syngeneic BMT. However, there is a paucity of information regarding immune recovery following T cell-depleted autologous/syngeneic BMT. We have developed a primate transplantation tolerance model in which rhesus monkeys were conditioned with total-body irradiation and extensively T cell-depleted autologous BMT and given a major histocompatibility complex-mismatched heterotopic cardiac allograft. This model provided an opportunity to study peripheral immune recovery following T cell-depleted autologous BMT. Limiting dilution analysis was used to quantify marrow T cells following depletion (2.8% to 25.6% marrow T cells predepletion, 0.00014% to 0.036% residual marrow T cells postdepletion). We found that (1) hematopoietic engraftment was prompt despite extensive marrow T cell depletion, (2) reconstitution of CD4+ helper T cells and CD8+ cytotoxic T cells were substantially delayed (6-12 months) compared with the recovery of CD8+ suppressor T cells, CD16+ NK cells, and CD20+ B cells, (3) distinction between CD8+ cytotoxic T cells and CD8+ suppressor T cells by the CD28 marker was critical in revealing the markedly discrepant recoveries of those subsets, and (4) immune reconstitution resembled that observed in recipients of T cell-depleted allogeneic and non-T cell-depleted autologous/syngeneic BMT, suggesting that the pattern of immune recovery following BMT is not substantially influenced by either allogeneic effects or the number of transferred T cells over a range of values.

Animals↗
PubMed1988

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants.

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.

Animals↗
PubMed1986

Culture and characterization of murine dendritic Thy-1+ epidermal cells.

Although numerous advances have been made in characterizing the phenotype, ontogeny, ultrastructure, and cytochemistry of the murine Thy-1+ dendritic epidermal cell (Thy-1+ EC), elucidation of its functional qualities has been hampered by the difficulty in preparing pure populations of these cells. We therefore sought to obtain expanded, purified populations of Thy-1+ EC using culture techniques. Since Thy-1+ EC are bone marrow-derived, density gradient enriched populations of freshly harvested epidermal cells (FH-EC) were placed in culture under conditions known or suspected to promote mitogenesis among leukocyte subsets. FH-EC prepared from truncal skin of C3H/HeN mice (Thy-1.2+) were cultured at 37 degrees C in 5% CO2 in complete medium (CM) of Eagle's Hanks' amino acid with 10% fetal calf serum, nutrients, and antibiotics at 10(6) FH-EC/well in 24-well culture plates. CM was supplemented with one or more of the following: concanavalin A (Con-A), interleukin-1/epidermal cell-derived thymocyte-activating factor (IL-1/ETAF), IL-2, IL-3, gamma interferon, indomethacin (IM), and anti-Thy-1.2 antibody. Media with appropriate supplements were changed every 2-3 days. Freshly isolated, enriched FH-EC contained 7-20% Thy-1+ EC (defined as brightly fluorescing cells readily distinguishable from weakly fluorescing keratinocytes), which also stained with antibodies directed against asialo GM1, Ly 5.1, and vimentin but did not stain with antibodies to other T cell-, B cell- or macrophage phenotypic markers. Analysis of 10 separate cultures revealed a 3- to 10-fold expansion of nonkeratinocyte Thy-1+ cells after 21 +/- 4 days in culture in CM supplemented with Con-A and IM, and 70-100% of viable cells after expansion were Thy-1+. Phenotypic analysis of expanded cells revealed the emergence in 10 separate cultures of one of two mutually exclusive distinct populations: one Thy-1+, asialo GM1+, L3T4- (natural killer phenotype) and the other Thy-1+, asialo GM1-, L3T4+ (T helper phenotype). Experiments designed to explain the emergence of an L3T4+ population suggest that phenotypic modulation occurred in vitro.

Animals↗
PubMed1986

Changes in mouse MHC expression during the cell cycle of Con A-treated spleen cells.

This study was directed at correlating the expression of class I MHC determinants with different segments of the cell cycle by using dual laser flow microfluorometry to measure levels of both DNA and cell surface H-2Kd or H-2Dd determinants for single cells. Con A-treated mouse spleen cells were identified as being in the G2/M or G0/G1 phases of the cell cycle on the basis of propidium iodide or Hoechst 33342 dye bound to DNA. Monoclonal anti-H-2 antibodies, indirectly fluoresceinated with goat anti-mouse IgG, were used to detect MHC determinants. The average level of both Kd and Dd determinants expressed by G2/M cells was about 1.6-fold higher than that expressed by G0/G1 cells. These observations indicate that the average-size G0/G1 and G2/M cells have the same apparent surface density of Kd and Dd determinants, insofar as we estimate that these cells differ in surface area by a factor of about 1.5. We also analyzed the expression of Kd and Dd determinants by measuring how they changed as a function of the intensity of forward light scatter from cells. For both G2/M and G0/G1 cells, changes in light scatter intensity were associated with parallel changes in levels of Dd and Kd determinants, indicating a common mechanism(s) that controls their cell surface expression.

Animals↗
PubMed1985

In vivo treatment with monoclonal anti-I-A antibodies: disappearance of splenic antigen-presenting cell function concomitant with modulation of splenic cell surface I-A and I-E antigens.

A single injection of anti-I-Ak antibody (AB) into H-2k mice resulted in abrogation of splenic antigen-presenting cell (APC) function for protein antigen-primed T cells or alloantigen-specific T cells. Spleen cells from anti-I-A-treated mice are not inhibitory in cell mixing experiments when using cloned antigen-specific T cells as indicator cells, thus excluding a role for suppressor cells in the observed defect. Also, nonspecific toxic effects and carry-over of blocking Ab were excluded as causes for the defect. Experiments with anti-I-Ak Ab in (H-2b X H-2k)F1 mice showed abrogation of APC function for T cells specific for both parental I-A haplotypes. In homozygous H-2k mice, anti-I-Ak treatment not only abrogated APC function for I-Ak-restricted cloned T cells but also for I-AekE alpha k-restricted cloned T cells. FACS analysis of spleen cells from anti-I-Ak-treated (H-2b X H-2k)F1 mice revealed the disappearance of all Ia antigens (both I-A and I-E determined), whereas the number of IgM-bearing cells was unaffected. The reappearance of APC function with time after injection was correlated with the reappearance of I-A and I-E antigen expression. In vitro incubation of spleen cells from anti-I-A-treated mice led to the reappearance of Ia antigen expression and APC function within 8 hr. Thus, it appears that B cells (as determined by FACS analysis) and APC (as determined by functional analysis) behave similarly in response to in vivo anti-I-A Ab treatment. We interpret these findings as suggesting that in vivo anti-I-A treatment temporarily reduces the expression of Ia molecules through co-modulation on all Ia-bearing spleen cells, thereby rendering them incompetent as APC. Such modulation of Ia molecules does not occur when spleen cells are incubated in vitro with anti-I-A antibodies. These results imply that a primary defect purely at the level of APC in anti-I-A-treated mice may be responsible for the observed T cell nonresponsiveness when such mice are subsequently primed with antigen.

Animals↗
PubMed1985

Quantitative measurements of the specificity and kinetics of conjugate formation between cloned cytotoxic T lymphocytes and splenic target cells by dual parameter flow cytometry.

The formation of conjugates between cloned anti-H-2Kb and Dd cytotoxic T lymphocytes (CTL) and splenic target cells has been studied by dual parameter flow cytometry. By varying effector-target combinations and by blocking with anti-MHC class I monoclonal antibodies, we found that the specificity of conjugate formation, in general, paralleled that expected from cytotoxicity studies; however, a significant number of "nonspecific" conjugates was always observed. As expected from previous studies, conjugate formation did not occur below 10 degrees C and was inhibited by cytochalasin B, EDTA, and anti-Lyt-2 antibodies. Conjugate formation followed first-order kinetics. The rate of formation of conjugates increased with temperature from 24 degrees to 37 degrees C; at 37 degrees C, the half-time was 1.4 min. After a 6-min lag period, lysis of target cells could be detected at 37 degrees C but not at 30 degrees C or below. Because target cell lysis proceeded during a period of time when the number of conjugates remained constant, considerable effector cell recycling must have occurred. Comparisons of the fluorescence emissions from conjugated effector or target cells with those from unconjugated cells demonstrated that nearly all conjugates contained one effector cell and one target cell, independent of the ratio of the two cell types in the original mix. Once formed, anti-H-2Kb conjugates were stable when diluted into medium alone, but rapidly disaggregated in medium containing either anti-Lyt-2 or anti-Kb monoclonal antibodies, both of which blocked conjugate formation. This finding suggests that conjugates are normally stabilized by intercellular bonds that are constantly breaking and reforming at the cell:cell interface, and that the antibodies disrupt the conjugates by preventing the reformation of broken bonds.

Animals↗
PubMed1984

Production of target-specific effector cells using hetero-cross-linked aggregates containing anti-target cell and anti-Fc gamma receptor antibodies.

Rabbit anti-2,4-dintrophenyl (DNP) antibodies or their F(ab')2 fragments were chemically cross-linked to the anti-mouse Fc gamma R monoclonal antibody 2.4G2 or to its Fab fragment. P388D1 cells were incubated with heteroaggregates between 2.4G2 and anti-DNP (anti-Fc gamma R X anti-DNP) and washed. The resulting cells lysed 2,4,6-trinitrophenyl chicken erythrocytes (TNP CRBC) in a hapten-specific manner. The lysis was inhibited by free hapten but was resistant to inhibition by immune complexes. Other cells coated with antibody heteroaggregates also mediated lysis of TNP-modified target cells. For example, mouse resident peritoneal exudate cells (PEC) lysed TNP CRBC and bacillus Calmette-Guérin-activated PEC lysed both TNP CRBC and TNP tumor targets. Human neutrophils, when incubated with heteroaggregates containing the anti-human neutrophil Fc gamma R antibody 3G8 and anti-DNP also lysed TNP CRBC and TNP-modified tumor cells. To test whether linkage to Fc gamma R was required for lysis, F(ab')2 fragments from the anti-KdDd monoclonal antibody 34-1-2 were cross-linked to anti-DNP F(ab')2 fragments. P388D1 cells (which express Kd and Dd) were then incubated with these heteroaggregates and washed, and their abilities to form conjugates and lyse TNP CRBC were compared with those of P388D1 cells treated with anti-Fc gamma R X anti-DNP. In both cases, P388D1 cells formed conjugates. However, only the cells treated with anti-Fc gamma R X anti-DNP mediated lysis to a significant extent. We conclude that heteroaggregates containing anti-Fc gamma R and anti-target cell antibodies can be used to create potent effector cells against red cell and tumor targets and that bridging of effectors with target cells directly to Fc gamma R on effector cells is required for lysis.

2,4-Dinitrophenol↗
PubMed1984

Filipin as a flow microfluorometry probe for cellular cholesterol.

The polyene antibiotic filipin, which forms specific complexes with 3 beta-hydroxysterols, displays spectral properties compatible with its use in flow microfluorometry (FMF). The purpose of this study was to test the suitability of filipin as an FMF probe for unesterified cellular cholesterol. The following experimental conditions appeared optimal for cells with an average unesterified cholesterol content of less than 3 nmol per 10(6) cells: 2 X 10(6) fixed cells (1-4% p-formaldehyde, 30 min, 21 degrees C) stained for 2-4 h with 100 micrograms/ml filipin and excited at 350.7/356.7 nm. Fluorescence emission (Em) was measured above 510 nm. Less suitable conditions involved excitation at 488 nm or using cells which had not been fixed. Fixation preserved the live-dead cell discrimination provided by forward light scatter measurements, so that dead cells could be excluded from the FMF analysis of cellular cholesterol. Under the above conditions FMF analysis of a variety of murine cell types showed that in all cases the fluorescence intensity of filipin-stained cells was clearly increased above autofluorescence levels of the unstained control cells. The increase in fluorescence signal in different filipin stained cell types correlated (P less than or equal to .001) with the cellular content of unesterified cholesterol determined by an independent enzymatic assay. The sensitivity of the FMF assay was in the femtomole (10(-15) ) range. Mixing experiments with cells of different cholesterol levels showed that the technique distinguishes cell populations with distinctive levels of unesterified cholesterol. We therefore concluded that filipin is a useful FMF probe for determining relative levels of unesterified cholesterol in cells.

Animals↗
PubMed1984

The measurement of specific cell: cell interactions by dual-parameter flow cytometry.

The Fc receptor-mediated aggregation of antibody-coated spleen cells with cells from the P388D1 mouse macrophage line was followed using a novel flow cytometric technique. P388D1 and spleen cells were directly labeled with green-emitting (fluorescein isothiocyanate) and red-emitting (substituted rhodamine isothiocyanate) fluorophores, respectively. They were mixed, incubated in suspension at 4 degrees C, and analyzed for aggregation with a dual laser flow cytometer. Unconjugated cells appeared as particles which were either red or green, while conjugates were detected as particles which were both red and green. Using this assay procedure, 5 X 10(4) cells were analyzed in 2-3 min for the percentages of conjugates, free spleen cells, and free P388D1 cells. Intercellular aggregation required both antibody on the spleen cells and free Fc receptors on the P388D1 cells; nonspecific aggregates accounted for 1% or less of the total particles analyzed. Measurements of the fluorescence distributions within conjugates indicated that the majority of conjugates contained a single P388D1 cell bound to 1-3 spleen cells, and that only heterophilic aggregation occurred. The flow cytometric technique described here should be applicable for the measurement of the initial events of intercellular aggregation in other systems as well.

Animals↗
PubMed1984

The mechanism of intercellular aggregation. I. The kinetics of the Fc gamma receptor-mediated aggregation of P388D1 cells with antibody-coated lymphocytes at 4 degrees C.

The formation of specific, heterophilic conjugates between cells from the P388D1 mouse macrophage line and antibody-coated mouse spleen cells was followed in cell suspensions at 4 degrees C by dual parameter flow cytometry. Intercellular aggregation in this system is mediated by the binding of the Fc portions of IgG antibodies on the spleen cells with Fc receptors (Fc gamma R) on P388D1. We show that the rate of aggregation reaches a plateau with increasing cell concentrations, suggesting that the initial collision between cells is not the rate limiting step of conjugate formation. The rates of aggregation are strongly dependent upon the cell surface densities of both Fc gamma R and antibody. In conjugates, however, only small fractions of available receptors or antibodies are utilized in bond formation. The rate-limiting step of aggregation, therefore, involves the formation of ligand-receptor bonds, and may be the diffusion of antibodies and receptors toward one another in small areas of intercellular contact. Inhibitor studies implicate microfilaments, but not microtubules, divalent cations, or energy-dependent processes as being important in aggregation. Finally, conjugates are stable when diluted into medium alone, but dissociate in media containing protein A, soluble immune complexes, or anti-Fc gamma R antibodies. This suggests that conjugates are stabilized by multiple intercellular ligand-receptor bonds, which constantly break and reform at the cell:cell interface, and that protein A, immune complexes, and anti-Fc gamma R disaggregate the conjugates by preventing the reformation of broken bonds.

Animals↗
PubMed1984

Quantitative analysis of Fc gamma receptors on murine spleen cell populations by using dual parameter flow cytometry.

The expression of Fc gamma R on subsets of mouse spleen cells was examined by dual parameter flow microfluorometry. B cells were detected by labeling them with antibodies against sIgM, sIgD, sIgG, or I-A; essentially all B cells expressed Fc gamma R. The number of Fc gamma R per cell on the sIgD+, sIgM+, and I-A+ cells averaged 2 X 10(4) receptors, and no correlation between the levels of expression of Fc gamma R and the B cell markers was evident. The sIgG+ B cells, however, expressed more Fc gamma R (8 X 10(4) receptors/cell) than sIgM+ and sIgD+ B cells. Fc gamma R on splenic macrophages were examined by double labeling spleen cells for Fc gamma R and Mac-1. The Mac-1+ cells (2 to 16% of the spleen cells) were 100% Fc gamma R+ and expressed threefold to fivefold higher numbers of Fc gamma R per cell than the sIgM+ or sIgD+ B cells. The Fc gamma R on T cells were studied on cells double labeled for Fc gamma R and Thy-1, Lyt-1, or Lyt-2. An average of 20% of the T cells expressed Fc gamma R and at least two subsets of Fc gamma R+ T cells were evident: Lyt-2- cells, most of which expressed intermediate (2 X 10(4) Fc gamma R/cell) levels of Fc gamma R, and Lyt-2+ cells, which expressed mainly high (8 X 10(4) Fc gamma R/cell) amounts of Fc gamma R. The levels of expression of Fc gamma R and sIgM increased dramatically in response to infection and were elevated in mice with genetic defects. We conclude that the level of Fc gamma R expression is a characteristic property of subsets of spleen cells from normal and infected mice.

Animals↗
PubMed1984

Characterization of murine lymphocyte IgE receptors by flow microfluorometry.

A flow microfluorometric technique has been developed to analyze IgE receptors on splenic and mesenteric lymph node mononuclear cells from BALB/c mice. Our data show that 1) the binding of DIBADL cross-linked IgE dimers to IgE receptors is specific in that it is inhibited by monomeric rat and mouse IgE but not by mouse or rabbit IgG or by the monoclonal anti-Fc gamma R antibody 2.4G2, and conversely, the binding of DIBADL cross-linked IgG dimers is inhibited by monomeric IgG or 2.4G2 but not by rat or mouse IgE; 2) the binding of IgE dimers is saturable on cells from uninfected and Nippostrongylus brasiliensis (Nb)-infected mice; 3) IgE dimer binding is detectable on most splenic B lymphocytes from uninfected and Nb-infected mice, but not on T lymphocytes from uninfected mice, and on few, if any, T lymphocytes from Nb-infected mice; 4) Nb infection causes a parallel increase in the percentages of B lymphocytes and cells expressing IgE receptors and Fc gamma R; 5) Nb infection leads to a marked increase in B lymphocyte IgE receptor expression, has little if any effect on IgE receptor affinity, and causes only minor changes in Fc gamma R expression; and 6) in vivo activation of B lymphocytes by a goat antibody to mouse IgD decreases IgE receptor expression considerably, but has a minimal effect on Fc gamma R expression. Thus, there are separate receptors for IgE and IgG on murine B lymphocytes, and the effect of Nb infection or anti-IgD treatment on their expression is different.

Affinity Labels↗
PubMed1983

Changes in cell-surface expression of MHC and Thy-1.2 determinants following treatment with lipid modulating agents.

Treatment of normal mouse spleen cells with lipid fluidity modulators changes the expression of cell-surface H-2 determinants. BALB/c spleen cells treated for 1 to 2 hr with cholesteryl hemisuccinate (CHS) displayed reduced levels of all tested H-2 determinants (H-2L, H-2K, and H-2D) as evaluated by flow microfluorometry and increased membrane lipid packing density as determined by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization. In contrast, decreasing membrane lipid packing density by phosphatidylcholine treatment decreased DPH fluorescence polarization and increased the expression of MHC determinants. The effects were selective in that expression of Thy-1.2 determinants was decreased by the latter treatment and not increased by CHS. The results are discussed in terms of passive modulation of antigenic expression.

Animals↗