Influence of tumor necrosis factor-alpha and interleukin-4 on the in vitro culture system for dendritic cells derived from cord blood CD34+hematopoietic precu rsor cells

Lei GU; Meifen XING; Xiaohui JI; Zhida SUN; Xiaofan YANG; Huijuan WANG; Mingshun ZHANG

ABSTRACT

BACKGROUND:

Blood stem cells (BSCs) are the most primitive cells in the immune system and can be differentiated into many kinds of cells. As the regulatory cells of immune response, dendritic cells (DCs) have been attracted more and more attention in the field of autoimmune diseases. Due to different resources of precursor cells, DCs have different cytokines, ideal cytokine matching,applying orders, and experimental cultured conditions. Furthermore, development, phenotypic expression, and mature degree are still different.

OBJECTIVE:

To investigate the influence of tumor necrosis factor- α (TNF- a ) and interleukin-4 (IL-4) on the culture system and provide improved method for inducing functional DCs in vitro, derived from cord blood CD34+ hematopoietic precursor cells (HPC).DESIGN, TIME AND

SETTING:

Observational study, which was performed in the Department of Microbiology and Immunology, Nanjing Medical University from March 2005 to November 2005.MATERIALS The cord blood was collected from neonatal umbilical cord in the 81 Hospital of Nanjing City. CD34 monoclonal antibody coated magnetic bead system (MACS) was provided by Miltenyi Biotec Company, Germany; human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF), human recombinant interleukin-4 (rhlL-4), and human recombinant tumor necrosis factor-α (rhTNF-α) by Pepro Tech Company, USA.

METHODS:

CD34+HPC were isolated and purified from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Then the cells were cultured with different culture medium which contained different combinations of cytokines GM-CSF and TNF-α (GT) or GM-CSF, TNF-α and IL-α (GTI) in order to be induced to differentiate into dendritic cells (DCs).The DCs derived from CD34+HPC were identified for their morphology and phcnotype by FACS and laser scanning con_focal fluorescence microscopy, and also for their abilities of inducing proliferation of allogenic T cells by 3H-TdR incorporation assay.

RESULTS:

The purity of selected CD34+ cells with MACS was more than 90%. DCs could be obtained from CD34+HPC by the culture in presence of GM-CSF and TNF-α or GM-CSF, TNF- o and IL-4. With the time of culture lasting, the cells expressed lower level surface antigen of CD34 and HLA-DR (P ＜ 0.05), and possessed the phenotypes of DCs characterized by higher expression of CD80, CD86, CD83 and CDIa. At 13-15 days, the cells possessed higher level of phenotypes of DCs compared to 7-9 days and 10-12 days. DCs induced with GTI culture system expressed higher levels of surface antigen CD80, CD86, CD83,CD 1 a and a lower level of CD 14 than those induced with GT culture system. DCs displayed typical morphology and property and expressed higher level surface antigen of CD86 and CD80, especially expressed CD86 when they were induced with proper cytokines of GM-CSF and TNF- α added at 0 hour, and of IL-4 added at 48 hours.

CONCLUSION:

DCs can be generated from CD34+HPC by proper culture system, the design of GM-CSF + TNF- α + IL-4(GM-CSF and TNF- α were added at 0 hour, IL-4 was added at 48 hours) is preferred.