Uwe Schauer

Detection of intracellular cytokines by flow cytometry

During the last years it has become increasingly clear that production of most cytokines is not confined to one cell type. Thus, a method to detect cytokines at the single cell level would be a helpful tool to study the contribution of different cells to cytokine production in heterogeneous cell populations. Recently, Sander et al. (1991) demonstrated that it is possible to detect intracellular cytokines by fixation with paraformaldehyde, permeabilization with saponin and subsequent indirect immunofluorescent staining using fluorescence microscopy. Here, we describe a modified method to increase the specific intracellular staining which enables us to detect IFN-gamma, IL-2 and IL-4 producing cells by single laser flow cytometry. The carboxylic ionophore monensin was used to interrupt intracellular transport processes leading to an accumulation of the cytokine in the Golgi complex. This resulting increase of the signal/noise ratio permitted us to detect weakly fluorescent cells such as IL-4 producing cells. While IL-4 was detected in approximately 1-3% of peripheral mononuclear cells from healthy donors, up to 30% of the cells produced IFN-gamma and nearly 50% IL-2 after phorbol ester and ionomycin stimulation. Microscopic and flow cytometric analysis showed a highly significant correlation. Using three-color flow cytometry it was possible to measure intracellular cytokines and cell surface markers simultaneously. Subpopulations of human T cells (e.g., CD4+ CD45R0-) producing a restricted cytokine pattern could be identified by cell surface staining and were characterized by their cytokine production. Consequently, there was no further need for cell sorting to determine cytokine producing subsets in heterogeneous cell populations. We have tested human T cell clones for intracellular cytokine production and found a high concordance to ELISA analysis of the supernatants. We conclude that detection of intracellular cytokines by flow cytometry is a rapid, easy and semiquantitative assay which may be used to study individual cells in heterogeneous populations as well as to screen homogeneous cells for their cytokine pattern. This method is particularly relevant in view of the accumulating evidence of the functional role that subsets of (T) cells may play in various diseases depending on the pattern of cytokines they produce.

Decreased frequency of interferon-γ– and interleukin-2–producing cells in patients with atopic diseases measured at the single cell level

BACKGROUND Recently, diminished interferon-gamma (IFN-gamma) and increased interleukin (IL-4 production in peripheral blood mononuclear cells (PBMCs) from atopic patients have been described by several groups, measured as total cytokine content in culture supernatants. These studies suggested a predominance of TH2-like cells producing large amounts of IL-4 in atopic patients. It is not clear whether the reported cytokine imbalances are the result of an alteration in the distribution of specific T-cell subsets or whether intrinsic dysregulation in cytokine production is a characteristic of atopic individuals. OBJECTIVE This study examined the production of IFN-gamma, and IL-2 in PBMCs from atopic patients at the single cell level with the use of freshly isolated lymphocytes. METHODS We recently described a flow cytometric assay in which three-color analysis was used to study the production of a cytokine of interest in a T-cell subpopulation defined by two cell surface markers. PBMCs from 23 atopic patients and 14 control subjects were stimulated with phorbol ester and ionomycin for 5 hours. PBMCs from seven patients and seven control subjects were also cultured with immobilized anti-CD3 antibodies for 24 hours. Cells were fixed, made permeable, and stained for intracellular cytokines in combination with cell surface markers CD3, CD8, and CD45RO. Cytokine-producing cells were analyzed by gating on T-cell subsets. RESULTS IFN-gamma-producing cells were significantly decreased (p < 0.05) in CD4+ T cells but not in CD8+ T cells of atopic patients. CD45R0+ and CD45R0-T cells showed a decreased proportion of IFN-gamma-producing cells (p < 0.05 and p < 0.01, respectively). IL-2 production was diminished in all T-cell subsets (p < 0.01). The number of IL-4-producing cells was not elevated, and such cells were exclusively found in the CD45RO+ T cells. Analysis of culture supernatants of sorted CD45RO+ T cells for IL-4 and IFN-gamma production confirmed these results. CONCLUSION Our findings provide evidence that a reduced IFN-gamma production in atopic patients is due to an intrinsic defect selectively found in the CD4+ T cells. Because IL-2 production was markedly decreased but IL-4 production was unchanged, our data demonstrate a deficiency in the ability of atopic T cells to produce TH1-like cytokines on stimulation with phorbol ester, ionomycin, or anti-CD3 monoclonal antibodies.

Respiratory syncytial virus induces prostaglandin E2, IL‐10 and IL‐11 generation in antigen presenting cells

Bronchiolitis caused by respiratory syncytial virus (RSV) infection is a major cause of hospitalization in children under 1 years of age. The disease characteristically does not induce protective immunity. However, a mononuclear peribronchiolar and perivascular infiltrate during RSV infection is suggestive of an immune‐mediated pathogenesis. Macrophages and dendritic cells (DCs) play an essential role in the initiation and maintenance of immune response to pathogens. To analyse interactions of RSV and immune cells, human cord blood derived macrophages and dendritic cells were infected with RSV. Both cells were found to be infected with RSV resulting in the activation of macrophages and maturation of dendritic cells as reflected by enhanced expression of several surface antigens. In the next set of experiments, generation of mediators was compared between cells infected with RSV, parainfluenza (PIV3) and influenza virus as well as ultracentrifuged virus free supernatant. Whereas the supernatant did not induce release of mediators, all three live virus infections induced IL‐6 production from macrophages and DC. Influenza virus infection induced predominantly IL‐12 p75 generation in DC. In contrast, RSV induced strong IL‐11 and prostaglandin E2 release from both macrophages and DCs. In addition, RSV but not influenza and parainfluenza virus induced a strong IL‐10 generation particularly from macrophages. Since IL‐10, IL‐11 and PGE2 are known to act immunosuppressive rather than proinflammatory, these mediators might be responsible for the delayed protective RSV specific immune response.

Levels of Antibodies Specific to Tetanus Toxoid, Haemophilus influenzae Type b, and Pneumococcal Capsular Polysaccharide in Healthy Children and Adults

ABSTRACT Antibody levels specific for capsular polysaccharides of Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) and for tetanus toxoid were measured in serum samples of 386 age-stratified subjects. The study group consists of healthy adult blood donors and hospitalized children undergoing elective surgery, excluding individuals with a history of infection. In children, anti-tetanus toxoid antibody levels displayed two peaks of 1.20 IU/ml (20.4 mg/liter) and 1.65 IU/ml (28.1 mg/liter) related to the schedule of routine childhood immunization in the first year and at 8 years of age. Eighty percent of the antibodies are of the immunoglobulin G1 (IgG1) isotype. For pneumococcal capsular polysaccharide (PCP), the specific antibody levels represent the acquisition of natural immunity. The initial concentration of 9.2 mg/liter was low in infancy (0.5 to 1 years of age) and remained low until 3 to 4 years of age (14.6 mg/liter). During this period PCP antibodies were almost 100% of the IgG2 subclass. Thereafter, IgG anti-PCP antibody titers increased steadily to adult levels (59.5 mg/liter). The data are intended to provide reference ranges to aid in the interpretation of specific antibody determinations in the clinical setting.

Measurement of intracellular cytokines

Abstract A newly developed technique enables intracellular cytokines to be detected in single cells. A recent workshop ★ brought together a group of exponents of this technique to discuss current experience with intracellular cytokine staining and the scope for further development.

Safety and efficacy of pimecrolimus in atopic dermatitis: a 5-year randomized trial.

BACKGROUND AND OBJECTIVES: Atopic dermatitis (AD) primarily affects infants and young children. Although topical corticosteroids (TCSs) are often prescribed, noncorticosteroid treatments are needed because compliance with TCSs is poor due to concerns about their side effects. In this longest and largest intervention study ever conducted in infants with mild-to-moderate AD, pimecrolimus 1% cream (PIM) was compared with TCSs. Methods: A total of 2418 infants were enrolled in this 5-year open-label study. Infants were randomized to PIM (n = 1205; with short-term TCSs for disease flares) or TCSs (n = 1213). The primary objective was to compare safety; the secondary objective was to document PIM’s long-term efficacy. Treatment success was defined as an Investigator’s Global Assessment score of 0 (clear) or 1 (almost clear). Results: Both PIM and TCSs had a rapid onset of action with >50% of patients achieving treatment success by week 3. After 5 years, >85% and 95% of patients in each group achieved overall and facial treatment success, respectively. The PIM group required substantially fewer steroid days than the TCS group (7 vs 178). The profile and frequency of adverse events was similar in the 2 groups; in both groups, there was no evidence for impairment of humoral or cellular immunity. Conclusions: Long-term management of mild-to-moderate AD in infants with PIM or TCSs was safe without any effect on the immune system. PIM was steroid-sparing. The data suggest PIM had similar efficacy to TCS and support the use of PIM as a first-line treatment of mild-to-moderate AD in infants and children.

Identification of eosinophils by flow cytometry

A flow cytometric method to identify and characterize eosinophils in lysed whole blood samples was established. A gating protocol was applied that in the first step uses the high autofluorescence and the high sideward scatter of eosinophils. In the second step, eosinophils were differentiated from neutrophils by lack of CD16 expression or alternatively presence of CD49d expression. Eosinophils purified by density gradient centrifugation (purity: 93% eosinophils contaminated with 7% neutrophils) were used to evaluate the technique. We were able to identify eosinophils added back to lysed whole blood samples and to identify partial degranulated eosinophils after treatment with secretory IgA and anti-IgA. In addition we were able to show that due to a large overlap of sideward scatter, the technique is applicable to purified normodense as well as hypodense eosinophils. In addition, there was a good correlation (r = 0.921, P < 0.0001) between the percentage of eosinophils determined by flow cytometry and microscopic evaluation in 81 patients. In patients with atopic dermatitis there was a reasonable correlation between a severity score (SCORAD) and the number of eosinophils determined by flow cytometry (R = 0.6107, P = 0017). Since the technique proved to be able to identify activated eosinophils bearing the CD69 early activation antigen, the relation between serum creatinine and CD69 expression on peripheral blood eosinophils was analysed showing a positive correlation (r = 0.4344, P = 0.016).

Safety of anti-IgE treatment with omalizumab in children with seasonal allergic rhinitis undergoing specific immunotherapy simultaneously.

Kamin W, Kopp MV, Erdnuess F, Schauer U, Zielen S, Wahn U. Safety of anti‐IgE treatment with omalizumab in children with seasonal allergic rhinitis undergoing specific immunotherapy simultaneously.
Pediatr Allergy Immunol 2010: 21: e160–e165.
© 2009 John Wiley & Sons A/S

A flow cytometric method for the detection of intracellular basic proteins in unseparated peripheral blood and bone marrow eosinophils

Eosinophils and their basic proteins play a major role in allergic disease and methods are required to monitor their expression in clinical situations. In this article we describe a flow cytometric method for the detection of intracellular eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in unseparated clinical samples. After fixation with parabenzoquinone and permeabilization with n-octyl-beta-D-glucopyranoside, the detection of intracellularly stored proteins was achieved using of monoclonal antibodies against ECP (EG1, EG2) and EPO in combination with an FITC-labeled second step antibody. Confocal microscopy was used to demonstrate the intracellular origin of the fluorescent signal. Fixation with parabenzoquinone was superior to a previously described protocol using paraformaldehyde, since it reduces non-specific binding of FITC to the basic proteins in eosinophils. Fixation and permeabilization do not alter the light scatter characteristics of eosinophils in contrast to other leukocytes and thus permit gating on eosinophils without prior purification. Furthermore, the procedure does not alter the detection of cell surface antigens on eosinophils and simultaneous measurements of surface antigens and intracellular proteins is possible. We have used different clinical samples (peripheral blood, bone marrow cells) to demonstrate differences in the expression of ECP and EPO. We conclude that the detection of intracellular eosinophil proteins by flow cytometry is a rapid, easy and semiquantitative procedure which may be used to study their expression in diseases where eosinophils are involved.

Differential response of human naive and memory/effector T cells to dendritic cells infected by respiratory syncytial virus

In vitro studies have contributed substantially to the understanding of immunopathology of respiratory syncytial virus (RSV)‐mediated disease. In the present study we compared the effect of RSV‐infected dendritic cells on the time–course of the primary and memory/effector T cell response in vitro. Cultures with uninfected dendritic cells known to elicit T helper 2 (Th2) responses and with polyinosinic‐polycytidylic acid (poly‐IC)‐stimulated dendritic cells known to elicit Th1 responses served as controls. At day 1 after stimulation there was a high proportion of interleukin (IL)‐2 and tumour necrosis factor (TNF)‐α‐producing T cells with no difference in number of producing T cells as well as concentration of secreted cytokines between RSV‐infected and control cultures. However, up to day 3 generation of IFN‐γ was reduced markedly. In addition, there was a reduced proliferation in RSV cultures. At day 7 the RSV‐treated cultures showed a preponderance of IL‐4 generation. At days 21–24, after three rounds of restimulation, memory/effector T cells matured under the influence of RSV were still not fully polarized but in contrast to the primary response displayed a predominance of Th1 cytokines. Contact with RSV‐infected HEp‐2 cells inhibited proliferation of T cells; memory effector T cells were less sensitive to contact inhibition than naive T cells. In addition, RSV inhibited the stimulated rearrangement of cortical actin more effectively in naive compared to memory T cells. In summary, we have shown that RSV infection of dendritic cells has a distinct modulatory effect on the primary response and a less pronounced effect on the memory response.