Flow cytometric analysis and modeling of cell-cell adhesive interactions: the neutrophil as a model.

The immune function of granulocytes, monocytes, lymphocytes, and other specialized cells depends upon intercellular adhesion. In many cases the molecules mediating leukocyte cell adhesion belong to the Leu-CAM superfamily of adhesive molecules. To elucidate the events of homotypic aggregation in a quantitative fashion, we have examined the aggregation of neutrophils stimulated with formyl peptides, where aggregate formation is a transient reversible cell function. We have mathematically modeled the kinetics of aggregation using a linear model based on particle geometry and rates of aggregate formation and breakup. The time course was modeled as a three-phase process, each phase with distinct rate constants. Aggregate formation was measured on the flow cytometer; singlets and larger particles were distinguished using the intravital stain LDS-751. Aggregation proceeded rapidly after stimulation with formyl peptide (CHO-nle-leu-phe-nle-tyr-lys). The first phase lasted 30-60 s; this was modeled with the largest aggregation rate and smallest rate of disaggregation. Aggregate formation plateaued during the second phase which lasted up to 2.5 min. This phase was modeled with an aggregation rate nearly an order of magnitude less than that of the initial fast phase, whereas the disaggregation rate for this phase did not change significantly. A third phase where disaggregation predominated, lasted the remaining 2-3 min and was modeled with a four to fivefold increase of the disaggregation rate. The mechanism of cell-cell adhesion in the plateau phase was probed with the monoclonal antibody IB4 to the CD18 subunit of the adhesive receptor CR3. Based on these studies it appears that new aggregates do not form to a large degree after the first phase of aggregate formation is complete. However, new adhesive contact sites may form within the contact region of these adherent cells to keep the aggregates together.

In vivo behavior of neutrophils from two patients with distinct inherited leukocyte adhesion deficiency syndromes.

The selectins and the beta 2-integrins (CD11/CD18) mediate distinct adhesive interactions between neutrophils and endothelial cells. Selectins are believed to initiate binding by mediating neutrophil rolling, whereas beta 2-integrins are required for subsequent activation-induced firm sticking and emigration. In vitro evidence suggests that two endothelial cell selectins, P- and E-selectin, can mediate rolling by binding to the carbohydrate ligand sialyl-Lewisx (sLex) on neutrophil surface glycoconjugates. To test the relative contribution of selectins and beta 2-integrins in vivo we used intravital microscopy to study the behavior of neutrophils from two patients with distinct inherited leukocyte adhesion deficiency syndromes. Neutrophils from a patient suffering from CD18 deficiency showed normal rolling behavior but were incapable of sticking or emigrating upon chemotactic stimulation. Neutrophils from a second patient with a newly described adhesion deficiency had normal CD18 but did not express sLex. These neutrophils rolled poorly and also failed to stick in venules under shear force. Under static conditions, however, chemoattractant-induced sticking and emigration could be observed. This demonstrates that both selectin-carbohydrate-mediated initiation of adhesion and subsequent activation-induced beta 2-integrin engagement are essential for the normal function of human neutrophils in vivo.

Influence of acridine tracer dyes on neutrophil function.

A study was performed to elucidate the effect of two commonly used fluorescent dyes in in vivo microscopic studies, acridine orange (AO) and acridine red (AR), on the ability of phorbol myristate acetate (PMA)- or formyl peptide (fMLP)-stimulated human neutrophils to adhere to a bovine serum albumin matrix and to generate superoxide anions (SOX). Unlabeled stimulated human neutrophils showed 36 +/- 9% (PMA, 10(-7) M) and 11 +/- 7% (fMLP, 10(-7) M) adherence to the matrix. This adhesion was CD18 dependent as evidence by 98% and 92% reduction, respectively, when the anti-CD18 antibody IB4 was included. A dose-dependent inhibition of stimulated human neutrophil adhesion was evident after 30 min of in vitro dye labeling and the EC50 was approximately 70 micrograms/ml (AR) and 145 micrograms/ml (AO). SOX generation by PMA-stimulated neutrophils was unaffected up to 100 micrograms/ml AR and AO but was reduced by 40-60% at higher doses. Rabbit neutrophils labeled in vivo or in vitro with 100 micrograms/ml AR exhibited 41% and 61% lower SOX generation, respectively. The study indicate that neutrophil function, in terms of ability to adhere to a BSA matrix using CD11/CD18 integrins and to generate SOX upon stimulation, is reduced depending on the dose and choice of fluorescent dye. Caution should be exercised when using these compounds at high concentrations in studies of PMN function.

Endocytosis of beta 2 integrins by stimulated human neutrophils analyzed by flow cytometry.

Flow cytometry and fluorescently labeled monoclonal antibodies were used to investigate endocytosis of human neutrophil beta 2 integrins following cellular activation. CD18 initially present on the cell surface cycled in two phases after exposure to formyl peptide or platelet-activating factor. The first phase lasted 3 min at 37 degrees C; after a lag, CD18 was specifically internalized at approximately 20%/min. Subsequently a second phase was detectable consisting of exponential reduction of internal fluorescence with a half-time of approximately 2 min, representing probe reexpression. At peak endocytosis approximately 40% of CD18 was internalized. All of the internalized CD18 was associated with alpha M (CR3); no endocytosis of alpha L (LFA-1) was observed. When neutrophils were stimulated with phorbol esters or calcium ionophore, CD18 was internalized much more slowly (t1/2 = 5 min) and probe was not reexpressed. Endocytosis of CD18 may participate in regulating neutrophil adhesiveness, removing activated receptors, or permitting receptor recycling.

Effect of adenosine on the expression of beta(2) integrins and L-selectin of human polymorphonuclear leukocytes in vitro.

Adenosine has been shown to inhibit the adhesion of polymorphonuclear leukocytes (PMNL) to the vascular endothelium. Because the underlying molecular mechanisms have not been fully understood, the present study characterizes the effect of adenosine on the expression of adhesion molecules of human PMNL. When PMNL were activated by N-formyl-methionyl-leucyl-phenylalanine the number of cell surface beta2 integrins increased fivefold, whereas L-selectin molecules were completely shed. Activation-dependent numerical up-regulation Of beta2 integrins and shedding of L-selectin were inhibited by exogenously applied adenosine receptor agonists in a concentration-dependent fashion. The rank order of potencies of adenosine receptor agonists, measured by the agonists' half-maximal inhibitory concentrations, revealed that adenosine inhibited the numerical up-regulation of beta2 integrins and shedding of L-selectin most likely via an A2(a) receptor site. When extracellular concentrations of endogenously formed adenosine were enhanced by the nucleoside uptake inhibitor dipyridamole, up-regulation of beta2 integrins, and shedding of L-selectin was again inhibited. Both effects were reversed by the enzyme adenosine deaminase, which degrades active adenosine to inactive inosine, suggesting that endogenously formed adenosine may play an important role in the regulation of beta2 integrins and L-selectin of human PMNL.

Multiwell cap assay: a simple objective method for the assessment of leukocyte locomotion in vitro.

A simple method for evaluating leukocyte locomotion in vitro has been developed and validated for several chemoattractants. The multiwell cap assay (MWCA) comprises chambers constructed from readily available disposable plastics and is quickly assembled, permitting large experimental protocols. Leukocytes which have migrated through a micropore filter are recovered and counted electronically yielding a precise, objective result. Coefficients of variation are approximately 6%.

Use of donors sharing one genetic haplotype for bone marrow transplantation.

Matched sibling transplants enjoy over 95% survival of the grafting procedure, but are only available for 1:5 patients. A sibling sharing one genetic haplotype is today our next choice of donor (67% survival) faring better than other relatives (50% survival), providing total body irradiation (of the thymus) has been avoided. The latter, without increasing the attack rate (64%) of GvHD more than doubles the deaths (57% as against 27%) attributable to it. Rejection is avoided by (a) suicide of host responders to donor buffy coat; (b) Cyclosporin-A; (c) displacement induction; (d) a higher dose of marrow. Prevention of GvHD is essential, using either Cyclosporin-A or removing donor T-cells from marrow prior to infusion or, probably better, both. Autoblast immunisation should be further explored. Tolerization seems an active process, easier in the very young, and nonirradiation of the thymus is believed important. An assay to assess tolerization (to guide cessation of immunosuppressive measures) is badly needed. Selection of a donor whose lymphocytes can deal with intracellular infections of the host's fibroblasts is now possible. The required increased immunosuppressive measures appear to increase the risk of leukemic relapse, and perhaps should be first improved in the more cost-effective fields of inborn error transplants.

Predicting licensed nurse turnover in skilled long-term care--organizational characteristics, climate, and group characteristics.

Chambers gives us information on turnover that directors of skilled long-term care facilities will want to know. The study looks at organizational characteristics, employee group characteristics, and work climate, as they relate to nurse turnover.

Technical aspects of recording evoked otoacoustic emissions using maximum length sequences.

Our previous work has demonstrated the feasibility of recording evoked otoacoustic emissions (EOAEs) using maximum length sequence (MLS) techniques. New equipment has been constructed, using standard DSP boards and computers, which permits stimulation at much higher rates. The technical aspects of analogue-to-digital converter (ADC) requirements, stimulus problems, artefacts, signal-to-noise ratio (SNR) computations and MLS application are considered here and illustrated with data from the new system.

Double-blind trial of the use of transfer factor in the treatment of Crohn's disease.

We have undertaken a double-blind controlled trial of the use of transfer factor in Crohn's disease. Thirty-three patients with known Crohn's disease completed the trial in which half the patients had three injections of transfer factor and the other half were given saline. After six months there was no significant difference in the clinical condition of either of the two groups compared with before receiving treatment. There was also no difference in their in vitro lymphocyte function, although a number of patients exhibited altered responsiveness to skin testing with tuberculin or streptokinase/streptodornase. A signficant fall on Crohn's disease activity index score occurred over the initial 'acclimatising period' before the trial was started, probably related to overcoming initial introspection and the placebo effect of being part of a trial.

Neutrophil aggregation is beta 2-integrin- and L-selectin-dependent in blood and isolated cells.

Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.

Construction of a steric map of the binding pocket for cannabinoids at the cannabinoid receptor.

In order to gain information about the topology of the brain cannabinoid receptor (CB1), a Receptor Steric (RS) Map for cannabinoids at this receptor was calculated. The classical cannabinoids (-)-11-hydroxy-delta-9-tetrahydrocannabinol (K1 = 210 +/- 56 nM), (-)-9-nor-9-beta-hydroxy-hexahydrocannabinol (K1 = 124 +/- 17 nM), nabilone (K1 = 120 +/- 13 nM), and the non-classical cannabinoid, CP-55,244 (K1 = 1.4 +/- .3 nM) were used as template molecules. The RS map was obtained as the union of the van der Waals' volumes of only those accessible conformers identified by MMP2 calculations that were able to clear a region of steric interference at the CB1 receptor previously characterized by us [Reggio, P.H., Panu, A.M. and Miles, S. (1993), J. Med. Chem., 36, 1761-1771]. The utility of the RS Map was explored by screening the accessible conformers of the classical cannabinoid, cannabinol (CBN), (K1 = 3200 +/- 450 nM), for its ability to fit within the RS map. Only the global minimum energy conformer of CBN (53.2% abundance at 298K) was able to fit within the RS map. These results imply that one reason for the reduced affinity of CBN may be that only 53.2% of CBN molecules are shaped properly to fit in the binding pocket for cannabinoids at the CB1 receptor.

C5a- and tumor necrosis factor-alpha-induced leukocytosis occurs independently of beta 2 integrins and L-selectin: differential effects on neutrophil adhesion molecule expression in vivo.

We previously demonstrated in rabbits that various neutrophil chemotactic factors share an ability to induce recruitment of polymorphonuclear leukocytes (PMN) from bone marrow when administered intravenously (Jagels and Hugli, J Immunol 148:1119, 1992). In the study reported here, we investigated the effects of chemotactic factors on the expression of beta 2 integrins and L-selectin in vivo and the roles of these adhesionmolecules in the recruitment process. Leukocytosis was induced by infusion of either C5a (5 micrograms/kg), N-formyl-methionyl-leucyl-phenylalanine (f-MLP; 2.5 micrograms/kg), or tumor necrosis factor alpha (TNF-alpha; 100 ng/kg). C5a increased the expression of CD18 (the common subunit of beta 2 integrins) on PMN by nearly twofold and decreased levels of L-selectin by 50% within 15 minutes after administration. Levels of beta 2 integrins returned to baseline 2 to 3 hours after induction of leukocytosis. L-selectin remained depressed for more than 5 hours, demonstrating that shedding was induced in the recruited bone marrow leukocytes as well as in circulating PMN. In contrast to the response to C5a, TNF-alpha did not cause upregulation of CD18 or shedding of L-selectin. Levels of L-selectin were consistently increased 60 minutes after administration of TNF-alpha, coinciding with a rapid rise in the number of band-form PMN in the circulation. Intact IgG and F(ab)2 forms of the anti-CD18 monoclonal antibody IB4 or the anti-L-selectin antibody DREG-200 were administered intravenously 15 minutes before induction of leukocytosis by the chemotactic factors. Neither IB4 nor its F(ab)2 fragments blocked leukocytosis induced by C5a, f-MLP, or TNF-alpha. DREG-200 also did not block leukocytosis induced by f-MLP, C5a, or TNF-alpha. These results suggest that leukocyte emigration from the bone marrow into the circulation proceeds through interactions distinct from those involved in neutrophil chemotaxis and diapedesis. Shedding of L-selectin from C5a-recruited bone marrow leukocytes demonstrates activation of these cells in the recruitment process and may reflect a potential mechanism for their release. The dissimilar effects of C5a and TNF-alpha on expression of adhesion molecules may result from distinct stimulatory pathways and suggests differential activation states for cellular recruitment by these inflammatory factors.

Timing of cyclosporin-A therapy for abrogation of HVG and GVH responses in rats.

Treatment with cyclosporin A was most effective in abrogating popliteal-lymph-node enlargement induced by host-versus-graft and graft-versus-host reactivity in rats when started before injection of donor-strain lymphocytes. Popliteal lymph-node enlargement was never completely abolished, and splenic lymphocytes from recipients treated with cyclosporin A showed no significant reduction in their response to donor-strain lymphocytes in mixed lymphocyte cultures, suggesting that clonal deletion had not taken place. Mixed lymphocyte cultures also indicated that cyclosporin treatment had not reduced the antigenicity of recipient lymphocytes towards donor strain.

High mannose type N-linked oligosaccharides on endothelial cells may influence beta 2 integrin mediated neutrophil adherence in vitro.

We report herein on the role of N-linked oligosaccharide processing of endothelial cell surface proteins on the adhesion of neutrophils. Monolayers of human umbilical vein endothelial cells were treated for 24 h with deoxymannojirimycin (DMJ), an inhibitor of golgi mannosidase I, which results in changes in glycoprotein processing, and then incubated with neutrophils to examine their ability to adhere to the treated endothelial cells. Treatment with DMJ, which leads to accumulation of high mannose type oligosaccharides, resulted in a twofold increase in adherence of phorbol ester (PMA) activated neutrophils compared to attachment to untreated endothelial cells. This adherence was likely mediated by the beta 2 integrin, Mac-1, and could be specifically inhibited with monoclonal antibodies to ICAM-1 and to the integrin beta 2 subunit. Similarly, IL-1 treatment resulted in a beta 2 integrin mediated increase in neutrophil adherence to the DMJ treated endothelial cells in a dose dependent manner. However, the IL-1 induced adherence was not significantly inhibited by the anti-ICM-1 antibody, thus, suggesting the presence of other inducible components on the endothelial cell surface. Our results demonstrate that alterations in glycosylation of N-linked oligosaccharides, resulting in the synthesis of high mannose type sugars on molecules that may interact with the beta 2 integrins, leads to an increased adherence of PMA activated neutrophils to endothelial cells.

Human immune responses to herpes simplex virus, varicella-zoster and cytomegalovirus in vitro.

The cell principally responsible for lymphocyte proliferation to herpes simplex virus (HSV), varicella-zoster (VZ) and cytomegalovirus (CMV) has been shown to be a T cell of helper phenotype. Lymphocytes from a proportion of proliferation-positive normal individuals produced anti-viral antibody in vitro. Although in some cases, and at some time-points, the antibody was specific for the priming virus, in others, antibodies to more than one virus were detected. Similarly, some T-cell clones proliferated specifically to the priming virus, whereas others were not specific for the virus used in the priming culture. Two clones helped the production of HSV-specific antibody, one by autologous, the other by both autologous and allogeneic non-T cells.

Expression of beta 2-integrins and L-selectin on polymorphonuclear leukocytes in septic patients.

Adhesion molecules on polymorphonuclear leukocytes (PMNL) play an important role in nonspecific defense mechanisms directed at invading microorganisms. When local infection, however, cannot be controlled, a systemic inflammatory response syndrome (SIRS) ensues which may progress to septic shock and multiple organ failure, these being major determinants of the patient's outcome. In the present study, the expression of beta 2-integrins and L-selectin on blood PMNL was measured on subsequent days in patients with sepsis (n = 17) and in healthy volunteers (n = 15). beta 2-Integrins and L-selectin molecules were detected by flow cytometry, using the monoclonal antibodies IB4 (anti-CD18) and Dreg200 (anti-CD62L), respectively. Adhesion molecules were determined at baseline immediately after blood collection and also 45 min after incubation of cells in vitro at body temperature to allow for spontaneous regulation. In addition, PMNL were activated by receptor-dependent and receptor-independent stimuli to characterize stimulus-specific adhesion molecule expression. In parallel with the measurement of adhesion molecules, severity of sepsis was assessed by the Elebute score. The results demonstrate significant differences in the basal, spontaneous and stimulus-induced expression of adhesion molecules between healthy volunteers, survivors (n = 11) and nonsurvivors (n = 6). Moreover, when survivors and nonsurvivors with severe sepsis (Elebute score > 12) were compared, basal expressions of both beta 2-integrins and L-selectin were significantly lower in patients who did not survive. Thus, measurement of adhesion molecules on circulating PMNL may be useful to identify septic patients at high risk for lethal outcome.

Correlation of cytogenetic abnormalities with the outcome of patients with uveal melanoma.

BACKGROUND: Cytogenetic investigations of choroid and ciliary body melanomas have revealed that the majority of cases are characterized by recurrent clonal abnormalities involving chromosomes 3, 6, and 8. The authors sought to determine whether these abnormalities were associated with outcome. METHODS: Fifty-four patients who underwent enucleation for untreated uveal melanoma between 1988 and 1996 were subjected to complete cytogenetic analysis. The most recent follow-up data from the time of enucleation was obtained. Patient outcomes were divided into two groups: 1) alive with metastases or dead of disease, and 2) alive without known metastases or dead of other causes. The relative risk (RR) and 95% confidence interval (CI) of a poor outcome were calculated for each chromosomal abnormality and clinical characteristic. RESULTS: Patients were followed for a median of 38 months. No patients were lost to follow-up. Abnormalities of chromosomes 3 and 8 were associated with a poor prognosis, but only when these two chromosomal abnormalities were present together (RR = 4.1, 95% CI = 1.7-9.9). The presence of a chromosome 6 abnormality was predictive of a good outcome (RR = 0.2, 95% CI = 0.1-0.8), even in the presence of chromosome 3 and 8 abnormalities. The only clinical factor associated with a poor outcome was the presence of extrascleral extension. CONCLUSIONS: In this group of patients with large posterior uveal melanomas, the concurrent presence of cytogenetic abnormalities of chromosomes 3 and 8 was associated with a poor outcome. An abnormality of chromosome 6 appeared to have a protective effect. These data suggest that cytogenetic analysis may provide prognostic information on patients with uveal melanoma and that further investigation of the contributions of these chromosomal aberrations to disease progression is warranted.