Immune phenotype and intracellular cytokine production of peripheral blood mononuclear cells from postmenopausal patients with osteoporotic fractures.

A number of factors with known effects on bone turnover are also immune regulatory factors. Disturbances of bone remodeling thus may be a consequence of altered local immune reactivity. We therefore determined surface markers and intracellular cytokine production of peripheral blood mononuclear cells by four-color flow cytometry in 19 postmenopausal patients with established osteoporosis and a control group of 11 postmenopausal women without fragility fractures. No significant differences in bone mineral density as assessed by dual energy X-ray absorptiometry were observed between the two groups. The following surface markers and cytokines were studied: CD3, CD4, CD8, CD16, CD19, CD29, CD45RA, CD56, CD57, HLA-DR, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-13, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and granulocyte macrophage colony stimulating factor. In the fracture patients, the percentage of CD8+ cells co-expressing CD57 was increased (14+/-2 vs. 8+/-1%; p=0.03). Moreover, the proportion of CD8+ cells co-expressing TNF-alpha (47+/-5 vs. 33+/-4; p=0.05) and both TNF-alpha and IFN-gamma was significantly higher in the patients than the controls (41+/-6 vs. 22+/-3%; p=0.04). IL-1beta expression tended to be increased in monocytes from patients with established osteoporosis. Distinct subsets of CD8+ cells thus appear to contribute to the development of osteoporotic fractures.

Regulatory effects of 1alpha,25-dihydroxyvitamin D3 on the cytokine production of human peripheral blood lymphocytes.

We studied the possible regulatory effects of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] on cytokine production and differentiation of subsets of CD4+ [T helper 1 (Th1) and Th2] and CD8+ [T cytotoxic 1 (Tc1) and Tc2] lymphocytes at the single cell level. PBMC from healthy donors were cultured with or without 1alpha,25-(OH)2D3 for up to 21 days. On days 0, 7, 14, and 21, the percentage of cytokine-producing T lymphocytes was analyzed by intracellular cytokine detection with mAb and flow cytometry. Simultaneous staining for cell surface markers allowed discrimination of CD4+ and CD8+ T cell subsets. After culture with 1alpha,25-(OH)2D3 (10(-8) mol/L), no significant effects on the proportion of interferon-gamma (IFNgamma)- or interleukin-4 (IL-4)-producing cells were detected, whereas reduced frequencies of IL-2-producing cells in the CD4+ as well as in the CD8+ population were found. An increase in the low percentage of CD4+ and CD8+ T cells producing the Th2 cytokine IL-13 was noticed. Most interestingly, IL-6-producing CD4+ and CD8+ T cells could only be detected in cultures with 1alpha,25-(OH)2D3, reaching a plateau after 14 days. The percentage of IL-6-producing T cells induced by 1alpha,25-(OH)2D3 after a given time period remained stable for at least 7 weeks. Studies of cytokine coexpression revealed that about 70% of IL-6-producing CD4+ and CD8+ cells were also positive for IL-2, but more than 90% were negative for IFNgamma, IL-4, or IL-13, respectively. This suggests that the IL-6-producing population does not match the Th1/Tc1-like (IFNgamma+) or Th2/Tc2-like (IL-4+ or IL-13+) subset. The influence of 1alpha,25-(OH)2D3 on cytokine production by lymphocytes is probably an important point of intersection between the endocrine and the immune system.

Cell surface characterization of T lymphocytes and allergen-specific T cell clones: correlation of CD26 expression with T(H1) subsets.

To evaluate the possibility of using surface molecules as markers for human T helper cell subsets, we studied the expression of surface molecules on T cell clones (TCCs) specific for the major birch pollen allergen, Bet v 1. No difference in the expression of the respective receptors for interferon-gamma (IFN-gamma), IL-4, IL-6, or IL-10 could be detected on T(H) subsets, nor did CD25 expression (IL-2R alpha-chain) differ significantly. However, high expression of CD26 antigen (dipeptidyl peptidase IV) correlated with a T(H1)/T(H0)-like phenotype, whereas T(H2)-like clones displayed a lower expression of CD26 antigen. Comparing cytokine production and CD26 expression simultaneously, we found a correlation between the IL-4/IFN-gamma ratio and the density of CD26 per cell. We could show that the amount of IL-4 secretion, and not of IFN-gamma secretion, was responsible for this correlation. To evaluate whether CD26 antigen expression is regulated by stimuli inducing a T(H1)- or T(H2)-like phenotype, peripheral blood mononuclear cells (PBMC) were cultured in the presence of IL-4, IFN-gamma, and IL-12, respectively. Incubation with IL-4 led to T cells with a T(H2)-like cytokine pattern and a significantly lower expression of CD26; IFN-gamma and IL-12 led to a T(H1) shift associated with an increased expression of CD26 on CD4+ T cells. By means of intracellular cytokine detection we analyzed expression of CD26 on CD4+ PBMC stimulated to produce IFN-gamma or IL-4 on a single cell level. All activated, cytokine-producing CD4+ T cells expressed CD26, but the increase in CD26 expression was higher in cells producing IFN-gamma. These data suggest that regulation of CD26 cell surface expression correlates with the production of T(H1)-like cytokines.