Establishment and Expression of Cytokines in a Theileria annulata-Infected Bovine B Cell Line

Abstract

This study aimed to establish a pure single-cell Theileria annulata-infected B cell line for the assessment of cytokine production in transformed and lipopolysaccharide (LPS)-stimulated cells. Several studies have aimed to identify cell surface markers in T. annulata-transformed cells; however, no information on cytokine production in these cells is available. To investigate the potential of the transformed cells to produce cytokines and their potential responses to antigen-stimulation, we purified mature B cells (CD21) from the whole blood of cattle experimentally infected with the T. annulata Kashi strain by magnetic separation. The purity and specificity of the established cell line was assessed by the identification of specific cell surface markers (CD21, IgM, and WC4) by flow cytometry analysis. The transcript levels of the cytokines IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL16, LTA, TGFB1, TNFA, IFNA, and IFNB in transformed, buparvaquone (BW720c)-treated cells, and antigen-stimulated cells were analyzed by quantitative polymerase chain reaction (qPCR) using cDNA from these cells. A T. annulata-infected bovine B cell line was successfully established with a purity of ~98.8% (CD21). IL4 and IL12A were significantly (p < 0.01) upregulated in the transformed cells. In BW720c-treated transformed cells, IL12B, TGFB1, and IFNB were significantly (p < 0.01) upregulated. Notably, no significant (p > 0.05) upregulation of cytokines was observed in LPS-stimulated transformed cells. Moreover, IL1A, IL1B, IL8, and IL16 were significantly (p < 0.01) upregulated in LPS-stimulated B cells. Our data signify the potential use of this cell line for cytokine production, observance of immunoglobulins, and production of an attenuated vaccine against tropical theileriosis.

Keywords: B cell line; Theileria annulata; antigenic stimulation; cytokines; transformation.

Figure 1

Schematic diagram for establishment of the Theileria annulata-transformed bovine B cell line. Here, N represents normal B cell culture, and T represents the infected cell culture wells used for transformation and proliferation for establishment of cell line.

Figure 2

Microscopic examination of T. annulata merozoites in red blood cells (RBCs) and macroschizonts in transformed B cells as well as normal cells stained with Giemsa observed under a compound microscope at 1000×. Here, (A,B) represent the normal RBCs and lymphocytes, respectively, while the arrows in (C,D) show the presence of merozoites in the RBCs of infected cattle and macroschizonts in the established cell line, respectively.

Figure 3

PCR amplification of 18S RNA with genus-specific primers for confirmation of piroplasm infection in experimental animals and the established cell line. Here, M represents the DNA marker (DL2000), lane 1 depicts the experimentally infected cattle with sporozoites, lane 2 depicts the transformed cells, lane 3 depicts the piroplasm free cattle, and lane 4 depicts the negative control.

Figure 4

Percentage of CD21⁺ cells in peripheral blood mononuclear cells (PBMCs) and analysis of their purity after magnetic separation. Here, (A) is the background control, (B) is the percentage of CD21⁺ cells in PBMCs, and (C) is the purity of magnetically isolated cells.

Figure 5

Percentage of CD21 in transformed B cells at the 3rd passage.

Figure 6

Percentages of B cell-specific surface markers (CD21, IgM, and WC4) present in transformed cells (6th and 10th generations), single-cell line clones (approximately 15th generation), and normal purified cells. The percentages of CD21 (98.8, 99.7, 98.0, 98.5, and 97%; mean ± SD of 98.4 ± 1.00), IgM (3.51, 0.64, 1.42, 0.99, and 2.83%; mean ± SD of 1.88 ± 1.23) and WC4 (0.51, 1.96, 0.71, 0.80, and 0.97%; mean ± SD of 0.99 ± 0.57) in five clones were determined. CD21 was present in mono- and polyclonal cell lines, while negligible percentage of IgM and WC4 were present.

Figure 7

PCR amplification of cytokine genes from TaBCs DNA and cDNA using specific primer pairs. Here, M represents the DNA marker (DL500), while lines 1–15 indicate IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12A, IL12B, IL16, TNFA, IFNB, IFNA, LTA, and TGFB1, respectively, amplified from DNA and cDNA harvested from of transformed B cells.

Figure 8

qPCR linearity measurements of bovine cytokines in cDNA from T. annulata-transformed B cells (TaBCs) and LPS-stimulated peripheral blood mononuclear cells (PBMCs). qPCR was performed on serially diluted (1/10) cDNA to detect and optimize the efficacies of various cytokine primers. These optimized primers were used to evaluate the transcript levels of cytokine in TaBCs, BW720c-treated transformed B cells, and LPS-stimulated normal B cells (nBCs) and TaBCs. The qPCR data were analyzed by regression analysis based on log values of sample quantity and threshold cycles (Ct) for each cytokine. The monitored cytokines and R² values of each regression line are shown.

Figure 9

Flow sheet diagram of samples collected from various cells types and the calibrators for cytokine production analysis.

Figure 10

A cytokine production network as well as interaction with themselves and other leukocytes is required for the stimulation of B cells to produce their own cytokines.

Figure 11

Transcript levels of cytokines in monoclonal T. anuulata-transformed B cells (TaBCs), BW720c-treated transformed cells, and LPS-stimulated TaBCs and normal B cells (nBCs). Here, *** p < 0.001, ** p < 0.01, * p < 0.05, and ns is not significant.

Figure 11

Transcript levels of cytokines in monoclonal T. anuulata-transformed B cells (TaBCs), BW720c-treated transformed cells, and LPS-stimulated TaBCs and normal B cells (nBCs). Here, *** p < 0.001, ** p < 0.01, * p < 0.05, and ns is not significant.