G. Halldén

A new membrane permeabilization method for the detection of intracellular antigens by flow cytometry

This article describes a new cell membrane permeabilization method for the detection of intracellular antigens by immunofluorescence staining and flow cytometry. The number of cells remained unaltered and no cell aggregation or loss of intracellular antigenicity was observed after this permeabilization treatment. A mixed leukocyte population from human peripheral blood was used in this study and the leukocytes were fixed and permeabilized, which permitted monoclonal anti-vimentin antibodies to reach intracellular antigens. The stabilization of cell membranes and preservation of intracellular antigenicity was achieved with paraformaldehyde fixation. This pretreatment prevents cell destruction and subsequent treatment with the detergent n-octyl-beta-D-glucopyranoside results in permeabilization of the cell membrane. The procedure does not alter the expression of cell surface antigens, which is of importance if phenotypic characterization of intracellularly stained cells is to be undertaken. Furthermore, this simple, rapid and reproducible technique makes it possible to detect and distinguish between different human peripheral blood leukocytes without prior purification steps. The leukocyte subpopulations remain well-separated and easily detectable by flow cytometry.

Altered expression of CD11b/CD18 and CD62L on human monocytes after cell preparation procedures

We have investigated the effect of different cell preparation procedures on the surface expression of CD11b/CD18 and CD62L on human monocytes. Both EDTA and heparin anticoagulated blood were used as sources for leukocytes. The monocytes were analysed by flow cytometry in a mixed leukocyte suspensions obtained after ammonium chloride mediated lysis and in mononuclear cell suspension prepared by density gradient centrifugation (Ficoll-Paque) performed both at 4 degrees C and at 20 degrees C. Monocytes from heparinized blood had a higher expression of CD11b/CD18 and a more pronounced inter-individual variation than monocytes from EDTA blood. Monocytes isolated by Ficoll-Paque had a higher degree of ex vivo activation by means of altered expression of CD11b/CD18 and CD62L compared to monocytes prepared by ammonium chloride mediated lysis. This was more pronounced when the isolation procedure was performed at 20 degrees C. Our findings indicate that monocytes prepared by ammonium chloride mediated lysis of EDTA blood and with the cell handling temperature kept at 4 degrees C are exposed to the smallest ex vivo modulation by means of receptor alteration. An awareness of ex vivo modulation by different cell preparation procedures is of importance especially when comparing the expression of functional receptors on leukocytes of disparate origin.

Not only Th2 cells but also Th1 and Th0 cells express CD30 after activation.

To investigate whether the CD30 molecule, expressed only by a minority of T and B cells, defines a subtype of T helper cells, Pityrosporum orbiculare‐specific CD4+ T cell clones were assessed for CD30 protein and gene expression. The clones were defined as Th1, Th0, and Th2 according to their cytokine mRNA profile detected by reverse transcription PCR (RT‐PCR). The kinetics of CD30 expression after OKT3 (anti‐CD3) stimulation was analyzed by flow cytometry, immunocytochemistry, and RT‐PCR. OKT3 activation induced a high expression of CD30 in cells of both Th1 and Th0 as well as Th2 type after 1‐3 days. A difference between the clones was noted in that the Th2 clones remained highly positive in CD30 expression, whereas expression in the other clones started to decline from day 3. These data indicate that CD30 is expressed in activated CD4+ T cells of all three subtypes, and that the expression is sustained in Th2 cells.

Enumeration of IFN-γ-producing cells by flow cytometry: Comparison with fluorescence microscopy

Abstract A new intracytoplasmic immunofluorescence staining to detect and quantify human interferon-γ (IFN-γ)-producing cells by means of flow cytometry is described. Mononuclear leukocytes, stimulated in vitro to produce IFN-γ, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic IFN-γ was demonstrated by indirect immunofluorescence using IFN-γ-specific mouse monoclonal antibodies. The staining exhibited a very characteristic morphology and was localized in the Golgi apparatus. An excellent agreement between the enumeration of cytoplasmic IFN-γ-positive cells by immunofluorescence microscopy and flow cytometry was noted. However, the latter has the advantage of a standardized control, is less labor consuming and is observer independent.

A flow cytometric method to measure the stimulated mobilization and the intracellular pool of the adhesion promoting glucoprotein Mac-1

The receptor for complement factor C3bi (Mac‐1 or CR3) belongs to a complex of leukocyte surface glucoproteins (CD11/CD18) that are essential for chemotaxis and adhesion of human polymorphonuclear leukocytes (PMN). Granulocytes can increase their surface expression of Mac‐1 upon stimulation and it is proposed that this depends on a rapid mobilization of an intracellular pool of Mac‐1. In the present study we describe a cell membrane permeabilization method that enables the detection of the intracellular pool of Mac‐1 in granulocytes by flow cytometry. The method is based on the use of the non‐ionic detergent n‐octyl‐beta‐D‐glucopyranoside (OG) to permeabilize the cell membranes of paraformaldehyde‐prefixed leukocytes. It is shown that fMLP (5 times 10‐7 M)‐treated cells expose 85% of the total detectable amount of Mac‐1 molecules on the surfaces. The method makes it possible to measure the total detectable pool, the efficiency of Mac‐1 mobilization and the in vivo expression of the receptor. This could be of value when evaluating the role of adhesion proteins in the inflammatory response.

MONOCYTE AND NEUTROPHIL ADHESION TO MATRIX PROTEINS IS SELECTIVELY ENHANCED IN THE PRESENCE OF INFLAMMATORY MEDIATORS

The authors investigated the time course of monocyte and neutrophil adhesion to fibronectin, vitronectin and albumin precoated culture wells, using mixed leucocyte populations from healthy blood donors. Moreover, the influence of chemotactic agonists on the adhesion properties as well as the quantitative expression of CD29, CD11b/CD18 and CD61 was analysed by flow cytometry. Different chemotactic agonists were used representing a classical chemotactic agonist (fMLP), and agonists with a preferential effect on monocytes (RANTES) and neutrophils (IL‐8), respectively. The authors found a gradual increase in monocyte and neutrophil adhesion to all three surfaces, reaching a plateau at 15 min of incubation. Adhesion to fibronectin was significantly higher at all time points (5, 15 and 60 min, respectively) compared with vitronectin and albumin in both monocytes and neutrophils. Neutrophil adhesion to vitronectin was significantly lower at 60 min compared with 15 min. Monocyte adhesion to albumin was increased by fMLP and RANTES and to vitronectin also by IL‐8. Neutrophil adhesion to albumin and vitronectin was increased by fMLP and IL‐8, but not RANTES. The adhesion to fibronectin was not altered by any of the chemotactic agonists used. The quantitative levels of CD11b/CD18, but not CD29 and CD61, was increased by fMLP, but not RANTES nor IL‐8. The authors conclude that the adhesion of human monocytes and neutrophils to vitronectin and albumin, but not fibronectin, is selectively enhanced by chemotactic agonists and may contribute to the selective accumulation of different leucocyte subsets at the inflammatory site.

In vitro effects of ethanol on polymorphonuclear leukocyte membrane receptor expression and mobility

The hampered inflammation and host defense seen in alcoholics may be due to impairment of functional responses of neutrophil polymorphonuclear leukocytes (PMN). We have shown that ethanol inhibits the oxidative metabolism of PMN induced by surface receptor dependent stimuli, such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and opsonized zymosan. Because the unresponsiveness might be due to reduced numbers of surface receptors, we assessed the expression of CR1, Fc-gamma, and fMLP receptors as well as membrane fluidity after treatment of PMN with ethanol in vitro. Ethanol impaired the induced expression of CR1 and fMLP receptors to 71% and 51% of control, respectively, but did not affect the resting level of CR1 nor Fc-gamma receptor expression. Furthermore, the mobility of cell membrane glycoconjugates was increased by ethanol. However, phagocytosis, a functional response dependent on membrane rheology, was unaffected. Because the results indicated an effect of ethanol on mobilization of receptors from intracellular stores, we assessed lactoferrin release, which was reduced to 59%. Thus, ethanol appeared to hamper the upregulation of PMN surface receptors or functional subsets of those stored in granules. Ethanol also increased the mobility of the cell membrane. These reactions were accompanied by reductions in the functional responses mediated by either class of receptors.

Differences in intracellular pool and receptor-dependent mobilization of the adhesion-promoting glycoprotein Mac-1 between eosinophils and neutrophils.

Recruitment of cells to an inflammatory site is a process that is selectively regulated. At an inflammatory site caused by bacterial infection, predominantly neutrophil accumulation is observed. This is in contrast to air lergic inflammation, where predominantly eosinophil accumulation occurs. Mac‐1 is an inducible adhesion molecule for both neutrophils and eosinophils. We examined the mobilization of this receptor on neutrophils and eosinophils after exposure to factors related to bacterial infections and allergic inflammation. We found more pronounced mobilization of Mac‐1 on neutrophils than eosinophils after exposure to N‐formylmethionyl‐ leucyl‐phenylalanine, lipopolysaccharides, and activated sera (C5a). There was no significant difference in Mac‐1 expression after exposure to aggregated immunoglobulin G. Incubation with interleukin‐5 (IL‐5) caused a significant increase of Mac‐1 expression on eosinophils but not on neutrophils. Neutrophils seem to respond to a greater extent than eosinophils to factors related to bacterial infections, whereas eosinophils respond better to IL‐5 associated with allergic inflammation. We measured the total pool of Mac‐1 to evaluate whether these differences could depend on the size of the intracellular pool. Eosinophils had a larger total pool of Mac‐1 than neutrophils. This finding increases the difference between eosinophils and neutrophils when relating the mobilized pool to the total pool. Stimulation with receptor‐ independent stimuli such as phorbol myristate acetate and ionomycin induced more pronounced mobilization of Mac‐1 on eosinophils, but no differences were obtained if the mobilized pool was related to their total pool. These results indicate that the difference in responsiveness depends on different receptor‐mediated signaling, since receptor‐independent stimulation resulted in relatively similar mobilization of the intracellular pool of Mac‐1.

Flow Cytometric Quantification of Lymphocyte Subpopulations and Immunoglobulin-containing Cells in Adenoid Tissue in Relation to Secretory Otitis Media and Age

This study was designed to identify differences in the immunological reactions in adenoid tissue between children suffering from chronic secretory otitis media (SOM) and control children without ear disease. Cell populations were identified using monoclonal antibodies and flow cytofluorometry to facilitate quantitative comparisons. A modification of the FOG method was developed to quantify lymphocytes with intracellular IgG and IgA. Immunological screening was done in the first part of the study. No significant differences were found between the groups regarding cells positive for CD3, CD4, CD8, CD20 or CD25. A significantly higher number of PCA-1 positive cells (presumably plasma cells) were found in the SOM group. The second part of the study concentrated specifically on cells containing IgG or IgA. No statistically significant differences in number of positive cells were found between the groups. When we related the percentage of positive cells to age, a statistically significant decrease with age for IgA-positive cells was found in the SOM group but not in the control group. This result supports the hypothesis that SOM is associated with an immunological reaction that influences immunoglobulin production in adenoid tissue.

PERIPHERAL BLOOD T-CELL RECEPTOR BETA -CHAIN V-REPERTOIRE IN ATOPIC DERMATITIS PATIENTS AFTER IN VITRO EXPOSURE TO PITYROSPORUM ORBICULARE EXTRACT

The yeast Pityrosporum orbiculare belongs to the normal cutaneous flora but is also considered to be one of the factors that may contribute to atopic dermatitis (AD). In the present study we investigated the possibility that P. orbiculare can act with superantigen activity in AD. P. orbiculare‐reactive T‐cell lines (TCLs) were obtained after stimulation of peripheral blood mononuclear cells (PBMC) with P. orbiculare extract. T‐cell receptor β‐chain V‐segment (TCRBV) usage was investigated using monoclonal antibodies and flow cytometry. We could not find any difference in TCRBV usage between AD patients (n = 10) and healthy controls (n = 5), either in fresh PBMC or in P. orbiculare‐reactive TCLs. Compared with their original PBMCs the P. orbiculare‐reactive TCLs showed a decreased usage of several TCRBVs, although increased usage of certain TCRBVs could be seen in some of the individuals. Further analysis of the CDR3‐length polymorphism exhibited a shift in CDR3‐length distribution, indicating oligoclonal expansion of T cells specific to different antigens in the P. orbiculare extract. In conclusion we have not found any evidence for superantigen activity in P. orbiculare extract, but our data support the importance of classical major histocompatibility complex (MHC)‐restricted allergens in P. orbiculare.