Cytokines and cell surface receptors as target end points of immunosuppression with cyclosporine A.

Targets of cyclosporine (CsA) were identified from an array of stimulated lymphocyte responses (sLR) comprising 34 stimulation conditions in whole blood from 3 normal human volunteers (NHV) containing clinically relevant CsA concentrations (0-1200 ng/ml) in vitro. In whole blood from 5 additional NHV, selected targets (intracellular interleukin-2 [IL-2], tumor-necrosis factor-alpha [TNF-alpha], and interferon-gamma [IFN-gamma]) were measured in phorbol myristate acetate (PMA)-ionomycin-stimulated T lymphocytes. Effect:concentration relationships were analyzed with E(max) pharmacodynamic (PD) equations and expressed as the concentration associated with one-half maximal inhibitory effect (EC(50)). CsA demonstrated a rich matrix of inhibitory effects on T cells (CD3(+)), B cells (CD19(+)), dendritic cells (DC) (CD11c(+)), and basophils (CD123(+)) but not on monocytes (CD14(+)) (n = 3). PD analyses suggested that the EC(50) of CsA (1) for IL-2 in CD3(+) cells in NHV (n = 8) was similar to the EC(50) demonstrated by us previously in CD4(+) cells from transplanted patients (n = 13) (EC(50) = 260 ng/ml vs. 249 ng/ml), (2) for each cytokine was different under identical stimulation conditions (TNF-alpha, 324 ng/ml; IFN-gamma, 504 ng/ml), and (3) was relatively constant for a given cytokine under different stimulation conditions (e.g., PMA-ionomycin or the staphylococcal enterotoxin B [SEB] superantigen). In conclusion, inhibition of cytokine targets by CsA is concentration dependent. Further, a given CsA concentration may produce similar inhibitory effects across different stimulation conditions. Measurement of cytokine target expression may, therefore, allow effect-controlled administration of CsA during clinical transplantation.

Experimentally induced recruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy.

Recent evidence suggests that the previously enigmatic cell type designated plasmacytoid monocytes can function as dendritic cells and contribute substantially to both innate and adaptive immunity. This cell type has previously been described only in bone marrow, blood, and organized lymphoid tissue, but not at effector sites with direct Ag exposure such as the mucosae. Plasmacytoid dendritic cells (P-DCs) matured in vitro can induce T cells to produce allergy-promoting Th2 cytokines; therefore, their possible occurrence in nasal mucosa during experimentally elicited allergic rhinitis was examined. Patients with silent nasal allergy were challenged topically with relevant allergen daily for 7 days. Biopsy specimens as well as blood samples were obtained before and during such provocation, and P-DCs were identified by their high expression of CD123 (IL-3R alpha-chain), together with CD45RA. Our results showed that P-DCs were present in low and variable numbers in normal nasal mucosa but increased dramatically during the allergic reaction. This accumulation concurred with the expression of the L-selectin ligand peripheral lymph node addressin on the mucosal vascular endothelium. The latter observation was particularly interesting in view of the high levels of L-selectin on circulating P-DC precursors and of previous reports suggesting that these cells can enter organized lymphoid tissue via high endothelial venules (which express peripheral lymph node addressin constitutively). Together, our findings suggested that P-DCs are involved in the triggering of airway allergy and that they are directed to allergic lesions by adhesion molecules that normally mediate leukocyte extravasation in organized lymphoid tissue.

A flow cytometric immune function assay for human peripheral blood dendritic cells.

CD11c+ and CD11c- (CD123+) dendritic cells (DCs) have been described in blood. Both cell types express high levels of HLA-DR and lack the lineage markers CD3, CD14, CD19, CD20, CD16, and CD56. These immunophenotypic properties were used along with analysis of activation-related surface antigens and intracellular staining of cytokines to characterize functional responses of these DC subsets to stimuli in whole human blood (WB). Samples from healthy donors were activated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate plus ionomycin (PMA+I). The only distinct response in CD11c- DCs was the expression of CD25 upon PMA+I activation. CD11c+ cells responded to LPS stimulation by producing high levels of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), and lower levels of IL-6, IL-1Ra, and IL-8 and an increased expression of accessory molecules (CD25, CD40, CD80, CD86, HLA-DR, and HLA-DQ). PMA+I activation of CD11c+ cells resulted in high levels of IL-1beta and lower levels of IL-8, IL-1Ra, and TNF-alpha and up-regulation of CD80, CD86, HLA-DR, and HLA-DQ. Our data support prior observations of functional differences between peripheral blood DC subsets and demonstrate the power of multiparameter flow cytometry to characterize the pleiotropic responses of these cells to various stimuli.

Flow cytometric ratio analysis of the Hoechst 33342 emission spectrum: multiparametric characterization of apoptotic lymphocytes.

The apoptotic response to various stimuli is an important part of immune regulation, and the ability to identify apoptotic lymphocytes within a complex population is a prerequisite to a more detailed understanding of its role in vivo, We described a flow cytometric technique which utilizes viable cells and enables simultaneous identification of apoptotic cells and analyses of immunophenotype, cell cycle progression, membrane integrity and light scatter properties. It is based upon analysis of two regions of the emission spectrum of the DNA-binding vital dye hoechst 33342. We established a precise correlation between the ratio of red to blue fluorescence emission and apoptosis based upon nuclear morphology and the presence of characteristic DNA degradation patterns. In human peripheral blood lymphocytes (PBL) and mouse thymocytes we incorporated light scatter properties, cell cycle stage, relevant cell surface immunophenotypic markers (CD25 or CD4) and CD8) and a marker of plasma membrane integrity (merocyanine 540) to enable multiparametric phenotyping of apoptotic cells. We show that staurosporine-induced apoptosis of ConA-stimulated PBL is not correlated with cell cycle stage but is selective for activated cells since the frequency of large, CD25+ cells is decreased by staurosporine. Dexamethasone and ionomycin differ in their ability to induce apoptosis selectively in murine thymocyte subsets. Dexamethasone kills a broad spectrum of the CD4/8 immunophenotypes with no selectively for cell cycle stage. Ionomycin selectively deplete CD4+8+ cells, especially those in the Go/G1 region of the cell cycle, and spared CD4-8+ cells. This technique is broadly advantageous for in vitro and ex vivo models of apoptosis in that it interrogates individual viable cells and correlates membrane and nuclear apoptotic changes with standard flow cytometric immunophenotyping.

Production of monoclonal antibodies using a secretion capture report web.

We describe a novel method for the production of monoclonal antibodies using a secretion capture report web (SCRW). Following HAT selection in bulk culture, individual hybridomas are encapsulated in biotinylated agarose drops. Antibody secreted by the hybridoma is captured within the agarose drop using an avidin bridge and biotinylated anti-mouse immunoglobulin. The secreted antibody is detected by a fluorescent reporter which can be either a second anti-mouse antibody or an antigen. The binding of the reporter can be quantitated and the desired hybridoma directly cloned by flow cytometry. Multiparameter (i.e., two-color) reporter analysis can also be used to selectively enrich and clone rare hybridomas secreting antibodies directed to unique epitopes. The method allows the characterization of thousands of clones per second and the isolation of hundreds of clones per day.

Secretion capture and report web: use of affinity derivatized agarose microdroplets for the selection of hybridoma cells.

A novel assay is described which allows the entrapment and detection of the immunoglobulin secreted from individual viable hybridoma cells using a secretion capture and reporter web (SCRW). By encapsulating the cells in agarose microdroplets which have been derivatized to create a fluorescent antigen-specific sandwich assay, flow cytometry can be used to identify and sort productive cells from a heterogeneous population. Using agarase, the cells can be recovered from the microdroplets and clonally expanded after selection. The assay has been used to reclone rare secretors from hybridoma cultures and to enhance the production of cultures with poor producers. The assay is easily generalized for the detection of any secreted protein for which specific antibodies or other ligands are available.

AT1 angiotensin receptors mobilize intracellular calcium in a subclone of NG108-15 neuroblastoma cells.

The effects of angiotensin II (AII) and related peptides on the mobilization of internal Ca2+ were studied in a subclone of NG 108-15 cells. The subclone, C1, was prepared by fluorescence-activated cell cloning using a rapid response kinetics and a large response magnitude following stimulation by AII as the selection criteria. Angiotensin I, AII, and angiotensin III (AIII) stimulated Ca2+ mobilization in the C1 cells in a concentration-dependent manner (1 nM-100 microM), yielding EC50 values of 437 +/- 80 nM (n = 4; slope = 1.6 +/- 0.3), 57 +/- 8 nM (n = 12; slope = 1.5 +/- 0.3), and 36 +/- 5 nM (n = 7; slope = 1.4 +/- 0.3), respectively. AIII was significantly more potent than AII (p less than 0.05). In contrast, Des-Phe8-AII, AII-hexapeptide (AII 3-8), and p-NH2-Phe6-AII (1-10 microM) were inactive as agonists. Although the effects of AII and AIII in C1 and parent NG108-15 cells were totally inhibited by the AT1 receptor-selective nonpeptide antagonist, DUP-753 (0.3-1 microM), the AT2-selective antagonists, EXP-655 and CGP42112A (1-10 microM), failed to block the effects of AII. DUP-753 (0.3-100 nM) produced dextral shifts of the AII-induced concentration-response curves and yielded an estimated affinity constant (pA2) of 8.5 +/- 0.2 (n = 16) using single-point analysis involving different concentrations of DUP-753. These data compared well with those obtained for the inhibition of AII-induced aortic contractions by DUP-753 (pA2 = 8.5) reported previously by others.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric analysis of internal calcium mobilization via a B2-bradykinin receptor on a subclone of PC-12 cells.

Single cell Ca2+ mobilization was studied by nonparametric, quantitative flow cytometry using a sort-selected subclone of PC-12 cells. The response of the parent PC-12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2-BK receptor agonists Lys-BK and Met-Lys-BK (10 pM-1 microM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1-BK receptor agonist Des-Arg9-BK (1 nM-1 microM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2-receptor antagonists D-Arg0-Hyp3-thienyl5,8-D-Phe7-BK and D-Arg0-Hyp3-D-Phe7 (0.1 nM-10 microM) in a concentration-dependent manner. Des-Arg9-Leu8-BK, a B1-receptor antagonist, failed to block the BK responses at 0.1-10 microM. The agonist/antagonist profile of the BK responses indicated that the B2-BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor-mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor-mediated second messenger generation at the single cell level.

Time window analysis and sorting.

Flow cytometric hardware and procedures were developed to continuously analyse and to sort a particular time window in a kinetic response. The technique uses balanced air pressure to drive a stimulus-bead mixture from a vial to a t-junction where it mixes passively with cells. The t-junction is distanced from the flow cell and air pressure regulated so that the stimulation occurs at a fixed and adjustable time before the cells are interrogated by the laser beam. Practical applications of the device demonstrate utility with cells whose responses are seen in seconds or minutes. The device is easily implemented on any sorting flow cytometer.

Detection of mRNA in flow-sorted cells.

Cells were sorted onto nitrocellulose filters which were saturated with a lysing cocktail designed to preferentially immobilize cellular mRNA. After washing, these filters were incubated with 32P-labeled specific DNA probes. We used the phorbol ester/lipopolysaccharide (PMA + LPS) co-induction of IL-1 mRNA and CD13 expression in U937 cells to demonstrate the specificity of the technique. In addition we used the abundant expression of c-fos in U937 to demonstrate linearity. IL-1 beta mRNA is readily discernable autoradiographically from as few as 5,000 PMA + LPS-induced cells sorted onto a filter. With liquid scintillation counting we demonstrate good linearity of the c-fos quantitation over the range of 1,000 cells to 60,000 cells per filter target. The technique is easily adapted to any sorting flow cytometer and should prove useful to help correlate any flow cytometric cell phenotype with specific mRNA abundance.

Use of a monoclonal antibody to classify neurons isolated from the head region of Hydra.

A mouse monoclonal antibody (JD1) to Hydra attenuata using the peroxidase-antiperoxidase (PAP) method revealed unipolar, bipolar, and multipolar sensory and ganglion cells in the head region of H. littoralis. Neurons isolated from macerated hypostomes and tentacles were classified according to the number of their cytoplasmic processes and the position of the cilium, when present, relative to the perikaryon. PAP-stained sensory cells had an apical ciliary cone, whereas ganglion cells did not. Neurons with cytoplasmic processes longer than 50 microns stained faintly, whereas those with processes shorter than 50 microns in length stained mainly dense brown. Unipolar neurons had an oval, crescent, round, or elliptic perikaryon with a single short axon. The perikaryal shape of bipolar neurons varied from round to tall triangular, short triangular, crescent, oval, or elliptic with two oppositely directed symmetric or asymmetric processes. Asymmetric processes were present in a bipolar sensory cell with a long apical cilium typical of gastrodermal sensory cells. One type of bipolar ganglion cell had a short perikaryal cilium. Another type had neurites longer than 50 microns. We found seven morphological variations of multipolar neurons, including one with an apical knob, two with a short perikaryal cilium, two with cytoplasmic loops near the perikaryon, one with perpendicular processes projecting from the major neurites, and one with a branched process longer than 50 microns opposite a tangled mass of neurites.

Spermatogenesis in Hydra oligactis. I. Morphological description and characterization using a monoclonal antibody specific for cells of the spermatogenic pathway.

A morphological description of cells participating in sperm formation in Hydra oligactis males using a maceration procedure is presented. These descriptions are corroborated by the use of a monoclonal antibody, AC2, that binds to both a subpopulation of interstitial cells that appears to participate exclusively in gamete formation, and to all the gamete-differentiation products, including sperm intermediate cells, spermatids, and sperm. Use of the antibody as an interstitial cell marker has allowed an analysis of the behavior of the gamete-precursor (AC2+) subpopulation of interstitial cells during the asexual state and the early stages of gamete formation, when no differentiating sperm intermediates are present. The results indicate there is a gamete-producing subpopulation of interstitial cells which is present in low numbers in asexual males and undergoes extensive growth following the onset of spermatogenesis to give rise to sperm intermediate cells, and, eventually, the sperm. No input from the AC2- interstitial cells is required to account for this growth or subsequent sperm production. We speculate that the AC2+ interstitial cells may represent a unique subpopulation which is developmentally restricted to sperm production.

Light and electron microscopic localization of a monoclonal antibody in neurons in situ in the head region of Hydra.

A mouse monoclonal antibody to Hydra attenuata was used to demonstrate immunoreactive product in neurons in situ, in both whole mount and sectioned hypostomes and tentacles of H. oligactis and H. littoralis. Immunoreactive cells were concentrated around the mouth and scattered along the length of the tentacles. In the hypostome, nerve cells sent one or more processes orally and the others aborally but the processes were more distinctly stained in H. oligactis. A thin strand of five to six perihypostomal neurons was present close to the hypostome-tentacle junction. In the tentacles, neurons with long processes contacted up to five different batteries of nematocysts. Neural processes were associated with nematocyst batteries in three ways: 1) forming a perikaryal loop to encircle a centrally located stenotele, 2) branching at a distance from the perikaryon to contact a variety of nematocysts, and 3) terminal branching by one or more neurons with contacts on one to several nematocysts within a battery. Immunocytochemical localization of neurons in Hydra by light microscopy was correlated for the first time with electron microscopy. Peroxidase-antiperoxidase (PAP)-positive sensory cells were concentrated around the mouth opening. PAP-positive ganglion cells were predominant in the tentacles. Sensory cells were elongate or spindle-shaped (unipolar), triangular with two oppositely directed processes (bipolar), and multipolar (tripolar or tetrapolar) with one of the processes extending to the epidermal surface. Ganglion cells were either unipolar or bipolar or multipolar, with neurites paralleling the mesoglea and occasionally having processes abut on it.

A subset of cells in the nerve net of Hydra oligactis defined by a monoclonal antibody: its arrangement and development.

A monoclonal antibody, termed JD1, was generated that bound to a subset of the nerve cells in the hypostome and tentacles of Hydra oligactis. Using a whole-mount technique the spatial pattern of the subset of nerve cells and their processes could be clearly visualized using indirect immunofluorescence. The subset largely corresponds to the epidermal sensory cells. Using the same technique the development of the pattern during head regeneration and budding was examined. The appearance of the nerve cells coincides with the formation of both the tentacles and hypostome. When head regeneration does not occur, JD1+ cells do not appear suggesting the differentiation of JD1+ cells is an integral event in head formation dependent on antecedent patterning processes.

Supervision of epileptic patients taking phenytoin.

Serum phenytoin concentrations in outpatients and inpatients with epilepsy have been contrasted. Individual patients were receiving prescriptions ranging from 100 to 300 mg. of sodium phenytoin daily, but the two groups were comparable in respect of dosage, age, and sex distribution. Whereas the mean phenytoin concentration among 14 inpatients was 28 mug./ml. that among 15 outpatients was only 15.7 mug./ml. When the supervision of a further 12 outpatients was changed to include more frequent visits and regular provision of blood samples their mean serum concentration of phenytoin rose from 17.5 to 27.7 mug./ml.The results indicate that failure to follow dosage instructions is a factor of major importance among epileptic patients whose response to treatment is inadequate or erratic.

Azathioprine in rheumatoid arthritis.

The ability of azathioprine to reduce the corticosteroid requirements of patients with severe rheumatoid arthritis was tested under double-blind conditions against placebo. After 12 months a significant mean dose reduction of 36% was achieved without undue side-effects. This form of therapy promises to be an advance in the management of severe rheumatoid arthritis.