Christine Neumann

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.

House dust mite-specific T cells in the skin of subjects with atopic dermatitis: Frequency and lymphokine profile in the allergen patch test

The mechanism underlying positive patch tests with house dust mite-allergen, Dermatophagoides pteronyssinus (Der p), in patients with atopic dermatitis was investigated by isolating T cells from the test sites of two patients. Eighty-five T cell clones (TCC) were established from the epidermis and dermis of lesional skin by the limiting-dilution method with Der p and interleukin (IL)-2. With restimulation assays, 29 of 60 TCCs tested demonstrated specific proliferation; 85% were of the CD3+, CD2+, and CD4+ phenotype. Der p-specific T cells constituted 0.4% to 2.7% of lesional T cells, and they were more frequent in the skin than in the blood of the patients by one order of magnitude. The mitogen-stimulated lymphokine profile of 55 TCCs was assessed; 42% (11/26) of the allergen-specific TCCs secreted IL-4 but almost no interferon-gamma, as described for the Th2 subset of the mouse. Also, six selected TCCs supported IgE secretion by autologous lymphocytes. Only three of 26 allergen-specific, skin-derived TCCs demonstrated a Th1-like lymphokine profile. These results support the specific nature of Der p-induced patch test lesions in patients with atopic dermatitis, and the results demonstrate also that a considerable proportion of lesional T cells are allergen-specific, IL-4-producing T cells that are capable of enhancing IgE production.

Rapid quantitation of proinflammatory and chemoattractant cytokine expression in small tissue samples and monocyte-derived dendritic cells: validation of a new real-time RT-PCR technology

The analysis of cytokine profiles plays a central part in the characterization of disease-related inflammatory pathways and the identification of functional properties of immune cell subpopulations. Because tissue biopsy samples are too small to allow the detection of cytokine protein, the detection of mRNA by RT-PCR analysis is often used to investigate the cytokine milieu in inflammatory lesions. RT-PCR itself is a qualitative method, indicating the presence or absence of specific transcripts. With the use of internal or external standards it may also serve as a quantitative method. The most widely accepted method is quantitative competitive RT-PCR, based on internal shortened standards. Recently, online real-time PCR has been introduced (LightCycler), which allows quantitation in less than 30 min. Here, we have tested its use for the analysis of cytokine gene expression in different experimental in vitro and ex vivo settings. First, we compared quantitative competitive RT-PCR with real-time RT-PCR in the quantitation of transcription levels of the CD4(+) cell-specific chemoattractant Interleukin-16 during the maturation of monocyte-derived dendritic cells, and found a good correlation between both methods. Second, differences in the amounts of IL-16 mRNA in synovial tissue from patients with rheumatoid arthritis and osteoarthritis as assessed by real-time RT-PCR paralleled differences in the level of IL-16 protein in the synovial fluid. Finally, we employed real-time RT-PCR to study the cutaneous expression of several cytokines during experimental immunomodulatory therapy of psoriasis by Interleukin-10, and demonstrate that the technique is suitable for pharmacogenomic monitoring. In summary, real-time RT-PCR is a sensitive and rapid tool for quantifying mRNA expression even with small quantities of tissue. The results obtained do not differ from those generated by quantitative competitive RT-PCR.

Nitric oxide inhibits the secretion of T-helper 1- and T-helper 2-associated cytokines in activated human T cells‘pa

Mechanisms regulating the balance of T‐helper 1 (Th1) and T‐helper 2 (Th2) immune responses are of great interest as they may determine the outcome of allergic and infectious diseases. Recently, in mice, nitric oxide (NO), a powerful modulator of inflammation, has been reported to preferentially down‐regulate Th1‐mediated immune responses. In the present study, we investigated the effect of NO on the production of Th1‐ and Th2‐associated cytokines by activated human T cells and human T‐cell clones. Cytokine secretion was measured in the presence of the NO‐donating agents 3‐morpholinosydnonimine (SIN‐1) and S‐nitroso‐N‐acetylpenicillamine (SNAP). Both NO‐donors markedly inhibited the release of interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), IL‐5, IL‐10 and IL‐4 by anti‐CD3 activated T cells. A preferential inhibition of Th1‐associated cytokines was not observed. Neither was nitrite found in the supernatants of activated T cells, nor was specific mRNA for inducible and constitutive NO synthase detectable, indicating that T cells themselves did not contribute to the observed effect of the NO donors. Costimulation with anti‐CD28 monoclonal antibodies (mAb) prevented SIN‐1/SNAP‐mediated down‐regulation of cytokine production only in part. In contrast, when T cells were stimulated by phorbol‐ester and ionomycin, they were refractory to SIN‐1‐induced inhibition of cytokine production. When SIN‐1 was added after the onset of anti‐CD3 stimulation, the inhibitory effect was found to be less pronounced, indicating that SIN‐1 may interfere with early signal transduction events. The addition of superoxide dismutase (SOD) and catalase did not restore the effects of SIN‐1, demonstrating that the inhibition of cytokines was due to NO and not to oxygen intermediates. Furthermore, 8‐Br‐cGMP‐mediated increase of intracellular cGMP caused the same pattern of cytokine inhibition as observed with SIN‐1 and SNAP. Using a single cell assay, these agents were shown to reduce the frequency of IFN‐γ‐producing T cells, suggesting that not all T cells are susceptible to SIN‐1/SNAP. However, cytokine production by purified T‐cell subpopulations (CD4+, CD8+, CD45RA+, and CD45RO+) was equally impaired by NO donors. In conclusion, in contrast to the murine system, our results do not provide evidence that NO preferentially inhibits Th1‐cytokine secretion of activated human T cells in vitro.

Double‐blind placebo‐controlled house dust mite control measures in adult patients with atopic dermatitis

Background Avoidance of allergens has been shown to be of benefit in patients with atopic asthma sensitized to indoor allergens. In atopic dermatitis, there is so far little information about the effect of house dust mite elimination strategies.

The treatment of patients with disseminated malignant melanoma by vaccination with autologous cell hybrids of tumor cells and dendritic cells.

Malignant melanoma has been shown to be susceptible to T cell-mediated immunity and, therefore, is a candidate for vaccination approaches. Clinical trials using dendritic cells (DC) loaded with peptides corresponding to tumor antigens are ongoing in several institutions, and some promising results have already been published. However, every single peptide-based vaccine can only be used in a patient with a given single HLA type, and this strategy is not appropriate for patients with rare HLA types or with tumors without defined antigens. A clinical pilot study in patients with disseminated melanoma refractory to standard therapy was initiated using a different approach. The authors generated autologous monocyte-derived DC and fused these DC with gamma-irradiated primary autologous tumor cells by incubation in polyethylene glycol. In previous experiments, the authors had shown that these fused cell products are potent inducers of a T-cell response in a mixed lymphocyte tumor cell culture. Seventeen patients were immunized with the cell product by s.c. injection in monthly intervals without any serious side effects. Of these patients, one had a partial response with decrease in size of all evaluable tumor manifestations. In one patient, some of the metastases were regressing despite an overall progressive disease, and one patient achieved disease stabilization for six months. In the responding patient, in parallel to tumor regression, circumscript hair depigmentation occurred. These data show, that a hybrid vaccine of DC and tumor cells can be safely applied and can induce tumor regressions, however, the clinical efficacy of the approach in its present form is insufficient.

Dynamic lymphoscintigraphy and image fusion of SPECT and pelvic CT-scans allow mapping of aberrant pelvic sentinel lymph nodes in malignant melanoma

To date, there are no reliable criteria to identify those patients with melanoma-infiltrated sentinel lymph nodes (SLNs) of the groin who might benefit from an extended lymphadenectomy, including the pelvic lymph nodes. We hypothesised that there are pelvic lymph nodes that receive lymph directly from the primary tumour, thus being at an increased risk for metastasis. In order to determine the frequency of radioactively labelled pelvic lymph nodes and the kinetics of their appearance, we introduce here a combination of dynamic lymphoscintigraphy, single photon emission computed tomography (SPECT) and image fusion of SPECT and pelvic Computed Tomography (CT)-scans. By dynamic lymphoscintigraphy and intraoperative gamma probe detection, superficially located inguinal SLNs (median 2 nodes) could be identified in all of the 51 patients included in this analysis. The histological search for micrometastases was positive in 16 patients (median Breslow thickness of the primary melanoma 2.5 mm). In 29 patients, SPECT and the image fusion technique were additionally performed. Radioactively labelled pelvic lymph nodes were detected in 20 individuals, 6 of them presenting aberrant pelvic SLNs that, on dynamic lymphoscintigraphy, had appeared simultaneously with the superficial SLN(s). Of the 6 patients in whom radioactive pelvic lymph nodes were excised together with the superficial SLN(s), only one had positive superficial SLNs. In this patient, the aberrant pelvic SLN proved to be tumour-positive. In 9 patients, there was no radiotracer uptake in the pelvic lymph nodes at all. Image fusion of SPECT and pelvic CT-scans is an excellent tool to localise exactly the pelvic tumour-draining nodes. The significance of radioactively labelled pelvic lymph nodes for the probability of pelvic metastases should be analysed further.

Engagement of the FcεRI Stimulates the Production of IL-16 in Langerhans Cell-Like Dendritic Cells

Preferential uptake and presentation of IgE-bound allergens by epidermal Langerhans cells (LC) via the high affinity IgE receptor, FcεRI, is regarded as an important mechanism in the induction of cutaneous inflammation in atopic dermatitis. Here, we show that activation of monocyte-derived LC-like dendritic cells (LLDC) through engagement of FcεRI induces the expression of IL-16, a chemoattractant factor for dendritic cells, CD4+ T cells, and eosinophils. We found that ligation of FcεRI on LLDC derived from atopic dermatitis patients that express high levels of FcεRI increases IL-16 mRNA expression and storage of intracellular IL-16 protein and enhances the secretion of mature IL-16 in a biphasic manner. An early release of IL-16 (peak at 4 h) is independent of protein synthesis, while a more delayed release (peak at 12 h) requires protein synthesis and occurs subsequent to the induction of IL-16 mRNA and intracellular accumulation of pro-IL-16. There was evidence that LLDC use caspase-1 to process IL-16, as inhibition of caspase-1, but not of caspase-3, partially prevented the release of IL-16 in response to ligation of FcεRI. In an in vivo model of IgE-dependent LC activation, the atopy patch test, positive skin reactions were also associated with the induction of IL-16 in epidermal dendritic cells. These data indicate that IL-16 released from LC after allergen-mediated activation through FcεRI may link IgE-driven and cellular inflammatory responses in diseases such as atopic dermatitis.

N-acetyltransferase 1 and 2 polymorphisms in para-substituted arylamine-induced contact allergy

Sensitization to arylamines such as p‐phenylenediamine is frequently diagnosed in patients with allergic contact dermatitis. Reactive metabolites of p‐phenylenediamine might be produced in the skin by O‐acetylation of N‐hydroxylamines catalysed by local N‐acetyltransferases (NATs). In this study, we tested whether genetic polymorphisms of NATs, which are known to affect enzyme activity, may influence the susceptibility to para‐substituted arylamine‐induced contact eczema. Using polymerase chain reaction and restriction enzyme analysis, the distribution of polymorphisms of NAT1 and NAT2 was investigated in 88 patients sensitized to para‐substituted aryl compounds and 123 healthy controls. NAT2 rapid acetylators, i.e. carriers of the NAT2*4 wild‐type allele, were more common in the contact allergy (44%) than in the healthy control group [30%; P = 0·042, odds ratio 1·9 (95% confidence interval, CI 1·05–3·27)]. Slow acetylators carrying the NAT2*5b/2*6a genotype were significantly less frequent among patients [13% vs. 38% in controls; P = 0·009, odds ratio 0·39 (95% CI 0·19–0·78)]. The carriage rate of the NAT1*10 allele, which is supposed to encode for a rapid NAT1 phenotype, was not significantly different between patients and controls [43% vs. 36%; odds ratio 1·5 (95% CI 0·88–2·68)]. Interactions between NAT2*4 and NAT1*10 were suggested by the increased frequency of the NAT2*4/NAT1*10 haplotype in patients (27%) compared with controls [15%; P = 0·039, odds ratio 2·1 (95% CI 1·04–4·04)]. As the NAT1 and NAT2 encoding genes are located in close proximity on chromosome 8p22, the latter finding could at least partly be due to genetic linkage. In fact, a linkage disequilibrium between NAT2*4 and NAT1*10 was observed in the contact allergy (P = 0·0025) and in the control group (P = 0·042). Our data indicate an association between the NAT2*4/NAT1*10 haplotype and contact sensitization to para‐substituted aryl compounds. Therefore, acetylation may either enhance contact sensitization or NAT2*4 and NAT1*10 might be linked to an unknown susceptibility factor.

Superficial Inguinal and Radical Ilioinguinal Lymph Node Dissection in Patients with Palpable Melanoma Metastases to the Groin - An Analysis of Survival and Local Recurrence

The present study addresses the question whether an extended ilioinguinal dissection as compared to an only superficial inguinal dissection improves survival and/or local tumour control after the appearance of palpable melanoma metastases to the groin. We retrospectively analysed the data of 104 patients with 69 ilioinguinal and 35 superficial inguinal dissections (median follow up 127 months). Prognostic factors of survival and groin recurrence were assessed using Kaplan-Meier estimation and Cox proportional hazards model. By multifactorial analysis, metastatic involvement of two lymph nodes or less was associated with a significantly better survival rate than involvement of >2 or pelvic nodes (p=0.0002). After radical ilioinguinal dissection, patients with extremity-located primaries had a better prognosis than patients with truncal primaries (p=0.03). Tumour infiltration of the ilio-obturator compartment was found to be an independent factor of poor prognosis (p=0.0009). The probability of recurrence in the dissected groin paralleled the number of positive nodes and significantly increased if intransits were observed (p=0.0002). The extent of surgery, Breslow thickness, epidermal ulceration, sex, age and adjuvant chemotherapy neither significantly influenced survival nor local control rates. In summary, when metastatic inguinal nodes become palpable, the presence of pelvic metastases indicates systemic disease. After therapeutic groin dissection, local recurrence and survival depend rather on regional tumour burden than on the extent of surgery.The present study addresses the question whether an extended ilioinguinal dissection as compared to an only superficial inguinal dissection improves survival and/or local tumour control after the appearance of palpable melanoma metastases to the groin. We retrospectively analysed the data of 104 patients with 69 ilioinguinal and 35 superficial inguinal dissections (median follow up 127 months). Prognostic factors of survival and groin recurrence were assessed using Kaplan-Meier estimation and Cox proportional hazards model. By multifactorial analysis, metastatic involvement of two lymph nodes or less was associated with a significantly better survival rate than involvement of > 2 or pelvic nodes (p = 0.0002). After radical ilioinguinal dissection, patients with extremity-located primaries had a better prognosis than patients with truncal primaries (p = 0.03). Tumour infiltration of the ilio-obturator compartment was found to be an independent factor of poor prognosis (p = 0.0009). The probability of recurrence in the dissected groin paralleled the number of positive nodes and significantly increased if intransits were observed (p = 0.0002). The extent of surgery, Breslow thickness, epidermal ulceration, sex, age and adjuvant chemotherapy neither significantly influenced survival nor local control rates. In summary, when metastatic inguinal nodes become palpable, the presence of pelvic metastases indicates systemic disease. After therapeutic groin dissection, local recurrence and survival depend rather on regional tumour burden than on the extent of surgery.