Intracellular cytokine repertoire in different T cell subsets from patients with sarcoidosis.

BACKGROUND: Pulmonary sarcoidosis is characterised by a mononuclear alveolitis with a predominance of CD4+ T cells and macrophages. We determined the intracellular expression of interferon (IFN)gamma, interleukin (IL)-2, tumour necrosis factor (TNF)alpha, IL-4, IL-5 and IL-10 in CD4+ and CD8+, naive and memory lymphocytes from blood and bronchoalveolar lavage (BAL) fluid using three colour flow cytometry. METHODS: Eighteen untreated patients with pulmonary sarcoidosis were evaluated and stratified according to whether they had acute or chronic disease. RESULTS: Significantly more T cells expressed Th1 than Th2 type cytokines in both BAL fluid and peripheral blood samples, regardless of clinical presentation. Significantly greater proportions of T cells secreted Th1 type cytokines in BAL fluid than in peripheral blood. Th1 type cytokines were more frequently expressed by peripheral and alveolar T cells in acute disease than in chronic disease. There were no significant differences between CD4+ and CD8+ T cells. Concerning naive and memory lymphocytes, significantly higher CD45RO:CD45RA ratios were found in BAL fluid than in blood, and increased expression of Th2 type cytokines was found in peripheral compared with alveolar memory T cells. CONCLUSIONS: Our data support the immunopathogenetic concept of Th1/Th2 imbalance and compartmentalisation in pulmonary sarcoidosis and suggest that the cytokine patterns change during the course of disease. Expression of Th2 type cytokines in memory lymphocytes is decreased in the alveolar compartment compared with peripheral blood.

Increased numbers of CCR5+ interferon-gamma- and tumor necrosis factor-alpha-secreting T lymphocytes in multiple sclerosis patients.

To determine the frequency of in vivo activated T(H)1 lymphocytes, T-cell subsets of 9 multiple sclerosis patients with active disease and 17 healthy controls were analyzed by immunostaining for CCR5, CD26, and their expression of interleukin-2, interferon-gamma, and tumor necrosis factor-alpha. The numbers of CCR5+ interferon-gamma- and tumor necrosis factor-alpha-producing T cells were significantly increased in the peripheral blood of multiple sclerosis patients. CCR5 expression may be a useful marker to identify effector cells in multiple sclerosis and could be used as a tool for monitoring disease activity.

Expression and kinetics of cytokines determined by intracellular staining using flow cytometry.

Cytokine production by human lymphocytes was assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. The production of interleukin 2 (IL2), interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was determined in T-helper (Th) and cytotoxic T-cells (CTL) as well as in naive and memory cells after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. Kinetic studies on IL2, IFNgamma and TNFalpha in lymphocyte subpopulations showed that TNFalpha was the first cytokine produced by T-cells. Production of this cytokine peaked at 2 h and declined rapidly thereafter. The peak of IL2 and IFNgamma production was at 8 h, and production of IL2 exceeded that of IFNgamma in T-cells at all times. IL2 production declined markedly after 8 h, while IFNgamma remained relatively stable for 24 h. IL2 and TNFalpha were mainly produced by Th cells while CTL primarily expressed IFNgamma. At all times a higher percentage of memory cells stained cytokine positive compared to naive cells and production of cytokines increased more rapidly in the memory cells. Naive cells produced primarily IL2, while memory cells expressed all the studied cytokines in substantial amounts. Kinetic studies between 1 and 24 h showed that 5 h was the optimal time point for evaluating the cytokines studied; hence normal values obtained from 50 healthy blood donors were evaluated after 5 h continuous PMA and ionomycin stimulation.

Polymorphisms in the tumor necrosis factor genes in Wegener's granulomatosis.

Two known di-allelic polymorphisms in the tumor necrosis factor (TNF) genes were investigated in 35 patients suffering from Wegener's granulomatosis (WG) in comparison to 111 healthy controls. The first polymorphism lay in the promotor region of the TNF-alpha gene at position -308, the second in the first intron of the TNF-beta gene. For both polymorphisms we could not find any statistically significant differences in allele frequencies between patients and healthy individuals. In the patient group the TNF 2/2 phenotype was slightly elevated, however (WG 5.7% vs. normal 1.8%). Investigating the clinical course of the disease according to TNF polymorphisms, TNF 1/1 patients were found to have a higher mean disease extension index (DEI) than TNF 1/2 individuals (TNF 1/1 10.5 vs. TNF 1/2 9.4).