Lucie Sedlackova

Cell surface and relative mRNA expression of heat shock protein 70 in human synovial cells

Heat shock proteins (Hsps) have been repeatedly implicated to participate in the pathogenesis of rheumatoid arthritis (RA). Methods: Herein, Hsp70 cell surface and mRNA expression were studied in human fibroblast-like synovial cells, dermal fibroblasts and peripheral blood leukocytes derived from 24 RA patients, who underwent synovectomy by using flow-cytometric analysis and real-time quantitative reverse-transcriptase polymerase chain reaction. For comparison, peripheral blood leukocytes of 17 healthy controls were tested. Results: Significantly higher Hsp70 membrane positivity was found on fibroblast-like synovial cells in RA patients (average 18.3%, median 16.5%) than on autologous and healthy control peripheral blood lymphocytes (RA patients: average 4.7%, median 2.9%, p = 0.002; healthy controls: average 6.0%, median 4.5%, p = 0.002) and/or autologous dermal fibroblasts (average 5.1%, median 4.3%, p < 0.001). Strong Hsp70 cell surface expression was also found on peripheral blood monocytes of RA patients (average 53.0%, median 58.1%) and healthy controls (average 49.4%, median 47.5%, p = 0.52). Peripheral blood granulocytes of healthy controls (average 41.8%, median 41.4%) showed significantly increased Hsp70 expression comparing with RA patients (average 10.7%, median 6.4%, p = 0.005). Significantly higher Hsp70 gene expression was observed in synovial cells of RA patients (average 2.04, median 1.7) when compared with autologous peripheral blood leukocytes (average 0.75, median 0.68; p < 0.001). However, the difference in Hsp70 gene expression between RA-derived synovial cells and healthy control peripheral blood leukocytes (average 1.69, median 1.64) was not observed (p = 0.83). We also found significantly lower relative gene expression in peripheral blood leukocytes of RA patients in comparison with healthy controls (p < 0.001). Interestingly, we found that Hsp70 gene expression in RA non-affected skin dermis gained from the operation wound was 3.7-fold higher in average (average 7.6, median 8.3) when compared to autologous RA-affected synovial tissue (p < 0.001); 10.1-fold higher in average when compared to autologous peripheral blood leukocytes (p < 0.001) and 4.5-fold higher in average comparing to control peripheral blood leukocytes (p < 0.001). Conclusion: Hsp70 gene expression in RA-affected synovial tissue is followed by Hsp70 cell surface expression on fibroblast-like synovial cells growing from RA synovial tissue. Hsp70 may be translocated to the cell surface from the cytosol and/or Hsp70 released from inflamed synovial tissue may be captured onto the membrane of synovial cells from the extracellular space via Hsp receptors. As a physiological response to potentially harmful enviromental stress factors, skin dermis produces higher levels of Hsp70 comparing to the cells of internal organs and tissues.

Heat-shock protein expression in leukemia.

Heat-shock proteins (Hsps) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, Hsp27, Hsp60, Hsp90α, and HspBP1 gene expression was investigated in human leukemia cell lines as well as in leukemia cells derived from patients with the onset of the disease. Hsp70 membrane expression and expression of Hsp27, Hsp60, Hsp70, Hsp90α, and HspBP1 genes were also tested in samples from leukemia patients. Relative Hsps gene expression was examined in human leukemia cell lines and also in patients, using real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Hsp70 cell surface expression was studied in patients with leukemia onset using flow cytometry. All tested cell lines showed significantly increased expression of Hsp60, Hsp90α, and HspBP1 genes compared with a cohort of healthy controls; additionally there was increased Hsp27 expression except for Jurkat and CCRF cells. Significantly higher gene expression of Hsp27, Hsp60, Hsp90α, and HspBP1 was observed in the peripheral blood of patients compared with bone marrow and healthy control samples, while Hsp70 expression was without any significant difference among cohorts. Hsp70 cell surface expression was found on defrosted and cultured leukemia cells but not on unprocessed biological samples from patients. Leukemia cells showed a heterogeneous pattern of Hsp gene expression among leukemia cell lines as well as in peripheral blood and bone marrow of patients.

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp-70-Derived 14-mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors

NK cells represent a potential tool for adoptive immunotherapy against tumors. Membrane-bound Hsp70 acts as a tumor-specific marker enhancing NK cell activity. Using flow cytometry the effect of in vitro stimulation with IL-2 or IL-15 alone or in combination with Hsp70-derived 14-mer peptide (TKD) on cell surface expression of NK activatory receptors (CD16, NKG2D, NKG2C, NKp46, NKp44, NKp30, KIR2DL4, DNAM-1, and LAMP1) and NK inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2, and NKR-P1A) in healthy individuals was studied. Results were expressed as the percentage of receptor expressing cells and the amount of receptor expressed by CD3−CD56+ cellular population. CD94, NKG2D, NKp44, NKp30, KIR2DL4, DNAM-1, LAMP1, NKG2A, and NKR-P1A were upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD. KIR2DL2/L3 was upregulated only by IL-15 and IL-15/TKD. Concurrently, an increase in a number of NK cells positive for CD94, NKp44, NKp30, KIR2DL4, and LAMP1 was observed. IL-15 and IL-15/TKD caused also cell number rise positive for KIR2DL2/L3 and NKR-P1A. Cell number positive for NKG2C and NKG2A was increased only by IL-2 and IL-2/TKD. The diverse effect of IL-2 or IL-15 w or w/o TKD on cell surface expression was observed in CD16, NKp46, and LIR1/ILT-2.

Heat shock protein gene expression profile may differentiate between rheumatoid arthritis, osteoarthritis, and healthy controls

Objective: Heat shock proteins (Hsps) have been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this work was to study Hsp mRNA and protein levels to determine whether they can be used to differentiate between RA, osteoarthritis (OA), and healthy controls. Methods: Hsp27, Hsp60, Hsp70, Hsp90α, and HspBP1 mRNA expression was analysed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 24 RA, 11 OA, and 21 healthy controls. Hsp70 and HspBP1 protein levels were measured in serum using an enzyme-linked immunosorbent assay (ELISA). Results: Hsp gene expression profiles differ significantly between inflammatory (RA) and non-inflammatory (OA) joint diseases, showing significantly increased Hsp27 and Hsp90α mRNA levels in RA synovial tissues. Up-regulated Hsp60 and Hsp90α together with down-regulated Hsp70 and elevated HspBP1/Hsp70 mRNA ratios can be used to differentiate between RA patients and healthy individuals through analysis of peripheral blood samples. Despite increased HspBP1 levels in RA sera, Hsp70 levels and the HspBP1/Hsp70 protein ratio remained identical in the RA patients and healthy individuals, which may contribute to the inhibition of Hsp70 anti-apoptotic activity. Conclusion: Hsp gene expression analysis can be implemented as a new diagnostic approach to facilitate differentiation between RA, OA, and healthy controls.

Fetal cells of mesenchymal origin in cultures derived from synovial tissue and skin of patients with rheumatoid arthritis

The transplacental cell transfer naturally takes place during pregnancy and occurs bi-directionally between the mother and fetus. Using real-time polymerase chain reaction (PCR) assay and sex determining region Y (SRY) gene as a marker, we examined the presence of male fetal cells in cell cultures derived from synovial tissues and skin dermis in women with prior pregnancy history suffering from rheumatoid arthritis (RA) who underwent synovectomy. Male DNA was detected in synovial cell samples derived from carpal, hip, metacarpophalangeal and metatarsophalangeal joints in five out of 13 (38.5%) patients with RA in a frequency range of 0.02-62.55 (mean 12.17) male cells per 10,000,000 total cells. SRY gene positivity was found as well in skin fibroblast cultures in four out of 10 (40.0%) RA patients in a frequency range of 3.26-43.47 (mean 15.42) male cells per 10,000,000 total cells, respectively. The difference in a frequency of fetal-derived male cells between both the cohorts did not achieve the statistical difference (p=0.77). We conclude that persisting male fetal cells are able to grow from non-inflamed tissues as well as from those which have many features characteristic of a stressed tissue. We conclude that persisting male fetal cells are also able to proliferate in cell culture since their presence was detected even in consecutive passages.

Analysis of cell surface and relative gene expression of heat shock protein 70 in human leukemia cell lines

Hsp70 was found to be expressed on the cell surface of leukemia cell lines compared with normal cells. Hsp70 cell surface and mRNA expression was studied in K562, Jurkat and CCRF-CEM human leukemia cell lines during several passages of in vitro culture. All the cell lines were positive for cell surface Hsp70 expression. However, increased mRNA expression was observed only in K562 cells where the expression was 9.88-fold higher on average compared with healthy controls. Jurkat cells, however, showed a significant negative correlation between cell surface and mRNA Hsp70 expression; high cell surface expression correlated with low mRNA expression in the Jurkat cell line.

Expression of heat shock protein 70 and NKG2D ligands in acute myeloid leukemia cell lines

Membrane-bound heat shock protein 70 (HSP70) was found to be tumor-specific and was proposed as a target for immunotherapy. In the present study, we analyzed cell surface and relative gene expression of HSP70 in cell lines established from patients with different acute myeloid leukemia (AML) subtypes, together with the expression of natural killer (NK) cell activation/inhibitory ligands. Materials and methods: Six AML cell lines were included in this study. The relative gene expression of HSP70 was analyzed using the real-time reverse-transcriptase polymerase chain reaction. Surface expression of HSP70 and NK cell ligands was analyzed using flow cytometry. Results: All cell lines overexpressed HSP70; however, its mRNA levels were not elevated. The expression of NKG2D activation ligands was heterogeneous. Conclusion: Our study is the first to describe long-term stationary cell surface expression of HSP70 in different subtypes of AML. Combined with the results of the gene expression experiments these data provide more evidence to the idea of a self-limiting mechanism for HSP70 expression.

Peripheral blood mononuclear cell responses to heat shock proteins in patients undergoing stem cell transplantation.

Abstract:  GVHD is a major complication after allogeneic SCT. Etiology of GVHD is multifactorial. Known role of hSPS in antigen presentation could suggest their potential role in the alloreactive process that leads to aGVHD. HSPS represent major immunodominant antigens in a wide spectrum of microbial pathogens. Bacterial and fungal colonization, infection and sepsis are frequent in immunocompromised patients with various malignant and non‐malignant diseases. We studied PBMC responses to recombinant human hsp60 (rh‐hsp60), rh‐hsp70 and Mycobacterium bovis hsp65 (M. bovis hsp65) in relation to aGVHD and infection in 34 pediatric patients with various lympho‐hemopoietic malignancies as well as non‐malignant disorders subjected to SCT. PBMC of patients before initiation of preparative regimen as well as after engraftment were stimulated with hSPS (1 μg/mL/well, 7‐day cultivation). PHA was used as a control of the stimulation ability. Cell responses were measured after the incorporation of 3H‐thymidin (pulsing with 1 μCi/well) and were expressed as stimulation indexes (SI). We demonstrated significantly high proliferative response to rh‐hsp60 as well as M. bovis hsp65 in a cohort of pretransplant patients with anamnestic and/or actual infection when compared with a cohort of patients without infection and healthy individuals. Strong PBMC cell responses to hSPS were found in patients who were at present colonized with Escherichia coli and Klebsiella pneumoniae or had previously K. pneumoniae infection with subsequent sepsis. Our findings support various studies dealing with immunodominant hSPS in connection with several pathogens and infectious diseases. Although no statistical difference for proliferative response to PHA was observed, PBMC responses against all tested hSPS comparing a cohort of patients with aGVHD and that with no sign of GVHD resulted in significantly lower SI for all tested hSPS in patients with aGVHD. Lower stimulation with hSPS during aGVHD might be explained by the stress‐induced upregulation of self‐hSPS synthesis that might lead to the inhibition of self‐hSPS reactive T‐cell response. Vice versa, we hypothesize that increased hsp‐specific stimulation may reflect the presence of protecting regulatory T cells preventing the development of Th1‐mediated diseases involving aGVHD.