Background The Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) is an inhibitory costimulatory molecule of the CD28 family, predominantly found on both regulatory T cells and activated T cells. It plays a crucial role in conveying immune tolerance by adjusting the threshold of T cell activation in inflammatory disorders and in regenerative processes such as bone healing. Human mesenchymal stromal cells (hMSC) are essential for fracture healing to reestablish skeletal integrity. They provoke immunomodulatory activities, but the mode of action is yet unknown. hMSC can be found in hypoxic fracture hematomas1, and we hypothesize these cells to support the termination of inflammation via CTLA-4 in order to facilitate bone fracture healing.
Objectives Aim of this work was therefore, to analyze hMSC with regard to their CTLA-4 expression and function.
Methods hMSC were isolated from bone marrow of patients undergoing total hip replacement. Isolation of human peripheral blood mononuclear cells (PBMC) was carried out by density gradient centrifugation from venous blood of healthy donors. The cells were stimulated for 48h with PHA (5μg/ml) or left untreated. Human MSC were characterized (i) by surface marker staining using flow cytometry (positive markers: CD13, CD44, CD90, CD105; negative markers: CD14, CD19, CD34, CD45) and (ii) via assessing their osteogenic and adipogenic differentiation potential. For the analysis of CTLA-4 expression in hMSC, cells were cultured under normoxic (∼18% O2) and hypoxic (<1.5% O2) conditions, respectively. The protein expression of CTLA-4 was analyzed by immunoblotting and flow cytometry whereas mRNA expression level was detected by quantitative PCR.
Results Isolated bone marrow - derived hMSC were positive for the expression of CD13, CD44, CD90 and CD105 and negative for CD14, CD19, CD34 and CD45. Human MSC character could be verified by osteogenic and adipogenic differentiation. The extra- and intracellular analysis of CTLA-4 in mitogen stimulated human PBMC demonstrated a CTLA-4 positive population of about 10,0%. On hMSC the CTLA-4 staining revealed a significantly positive shift (p=0,027). Western blot analyses confirmed the CTLA-4 expression by hMSC. hMSC cultured in normoxic conditions showed a significantly higher mRNA level of CTLA-4 than hMSC incubated in hypoxic conditions, but CTLA-4 mRNA was verifiable in both. In western blot and flow cytometry analysis the CTLA-4 expression did not alter under different oxygen availabilities. Altogether, we demonstrated for the first time that hMSC indeed express CTLA-4 on both mRNA and protein level. Hypoxia did not have a measurable effect on CTLA-4 expression of hMSC, merely the mRNA level decreased.
Conclusions We could clearly demonstrate the existence of CTLA-4 on hMSC. We assume the expression of CTLA-4 (i) to contribute to the immunomodulatory capacity of hMSC and (ii) to support the 'immune privileged' status of these cells. These hypotheses will be proven in further studies.
Kolar P, Gaber T, Perka C, Duda GN, Buttgereit F. Human early fracture hematoma is characterized by inflammation and hypoxia. Clin Orthop Relat Res. 2011, 469:3118-3126
Disclosure of Interest None declared