Different distribution of CD4 and CD8 T cells in synovial membrane and peripheral blood of rheumatoid arthritis and osteoarthritis patients.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are chronic diseases associated with morphological joint changes. Synovial membrane (SM) involvement was established for RA, but the data for OA are limited, because OA is usually regarded as noninflammatory disease. Changes in immune system in RA are not limited to joints, and the significant role of T cells of peripheral blood (PB) is not disputable. However, there is still an open debate about PB immunological profile in OA. Therefore, we decided to measure the distribution of CD4+ and CD8+ T cells, regarding CD28 expression, both in PB and SM of RA and OA patients, on the same day. Altogether, eleven RA patients, 11 OA patients and similar numbers of age-matched healthy controls were included into the study. Flow cytometry was used for T cells subpopulation distinguishing and quantification; monoclonal antibodies against CD3, CD4, CD8 and CD28 with different fluorochromes were used for stainings. The RA patients had significantly higher percentage of CD3+4+ cells in PB as compared to OA patients and relevant control group. Both within the CD4+ and CD8+ compartments, significantly lower percentages of cells bearing the CD28 marker were found in the PB of OA as compared to RA patients. The proportion of CD3+CD4+ cells in SM was dependent on age of OA patients, older OA patients had significantly higher value of their SM/blood ratio than RA patients. Older OA subjects were also characterized by higher values of the SM/blood ratio of both CD4+CD28+ and CD8+CD28+ subpopulations than RA or younger OA patients. In conclusion, in contrast to the traditional view of OA disease, our results give support to the hypothesis that OA may also (like RA) be a disease with a local immunological involvement

Expression of calpain-calpastatin system (CCS) member proteins in human lymphocytes of young and elderly individuals; pilot baseline data for the CALPACENT project.

Ubiquitous system of regulatory, calcium-dependent, cytoplasmic proteases – calpains – and their endogenous inhibitor – calpastatin – is implicated in the proteolytic regulation of activation, proliferation, and apoptosis of many cell types. However, it has not been thoroughly studied in resting and activated human lymphocytes yet, especially in relation to the subjects’ ageing process. The CALPACENT project is an international (Polish-Italian) project aiming at verifying the hypothesis of the role of calpains in the function of peripheral blood immune cells of Polish (Pomeranian) and Italian (Sicilian) centenarians, apparently relatively preserved in comparison to the general elderly population. In this preliminary report we aimed at establishing and comparing the baseline levels of expression of μ- and m-calpain and calpastatin in various, phenotypically defined, populations of human peripheral blood lymphocytes for healthy elderly Sicilians and Poles, as compared to these values observed in young cohort

Changes in the Expression of Transcription Factors Involved in Modulating the Expression of EPO-R in Activated Human CD4-Positive Lymphocytes.

We have recently described the presence of the erythropoietin receptor (EPO-R) on CD4(+) lymphocytes and demonstrated that its expression increases during their activation, reaching a level reported to be typical for erythroid progenitors. This observation suggests that EPO-R expression is modulated during lymphocyte activation, which may be important for the cells' function. Here we investigated whether the expression of GATA1, GATA3 and Sp1 transcription factors is correlated with the expression of EPO-R in human CD4(+) lymphocytes stimulated with monoclonal anti-CD3 antibody. The expression of GATA1, GATA3 and Sp1 transcription factors in CD4(+) cells was estimated before and after stimulation with anti-CD3 antibody by Western Blot and flow cytometry. The expression of EPO-R was measured using real-time PCR and flow cytometry. There was no change in the expression of GATA1 and GATA3 in CD4(+) lymphocytes after stimulation with anti-CD3 antibody. However, stimulation resulted in the significantly increased expression of the Sp1 factor. CD4(+) lymphocytes stimulated with anti-CD3 antibody exhibited an increase in both the expression level of EPOR gene and the number of EPO-R molecules on the cells' surface, the latter being significantly correlated with the increased expression of Sp1. Sp1 is noted to be the single transcription factor among the ones studied whose level changes as a result of CD4(+) lymphocyte stimulation. It seems that Sp1 may significantly affect the number of EPO-R molecules present on the surface of activated CD4(+) lymphocytes

Changes in the number of EPO-R molecules per cell, <i>EPOR</i> gene expression and Sp1 expression in CD4⁺ lymphocytes before and after stimulation with anti-CD3 antibody.

<p>The figure shows results from 3 different individuals, MFI – mean fluorescence intensity, RFU – relative fluorescence units.</p

Comparison of GATA1, GATA3 and Sp1 expression determined by Western Blot in isolated CD4⁺ lymphocytes before and after stimulation with anti-CD3 antibody.

<p>Figure A shows representative results. Figure B presents mean expression of GATA1, GATA3 and Sp1 in arbitrary densitometric units. Figures C, D and E present expression of GATA1, GATA3 and Sp1, respectively. Midpoints of figures present medians, boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data, *p<0.05, Friedman ANOVA and Post Hoc test.</p

Fluorescence analysis of EPO-R and Sp1 expression measured by flow cytometry in CD4⁺ lymphocytes before and after stimulation with anti-CD3 antibody.

<p>CD4⁺ cells were selected on the basis of forward and side scatter characteristics of lymphocytes, <i>Gate 1</i> (A, E) and expression of CD4 antigen in gated lymphocytes, <i>Gate 2</i> (B, F). Figures C and D present the expression of EPO-R and Sp1 in CD4⁺ cells (cells from <i>Gate 1</i> and <i>Gate 2</i>) before stimulation, figures G and H present the expression of EPO-R and Sp1 in CD4⁺ cells stimulated with anti-CD3 antibody for 48 hours, respectively. For individual samples, expression of EPO-R and Sp1 was estimated as mean fluorescence shift (black line) toward isotype control (gray histogram): δMFI = MFI of the population of interest – MFI of the appropriate isotype control. Quantitative fluorescence analysis assessing the number of EPO-R molecules per cell based on δMFI was performed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060326#pone.0060326-Lisowska1" target="_blank">[3]</a>.</p