Trauma patients account for about 25–35% of the patients visiting the emergency center, which takes a large proportion of deaths and disabilities among young age groups resulting in national losses due to the loss of economic ability.
In many countries, it is difficult to reduce on-site deaths even though trying to improve the level of primary care in hospitals and reduce complications that occur after primary treatment such as sepsis, multiple organ failure. It is important to maintain the homeostasis of trauma patients until surgical treatment in order to alleviate hyper-inflammatory and immune-paralysis conditions to prevent the occurrence of post-traumatic secondary complications, such as sepsis, multiple organ failure. This study was conducted because many patients died despite the proper treatment. The tendency of macrophages to differentiate THP-1 cells, which are human monocytic cell lines, is showing changes in macrophages that respond early in the event of various damage, such as trauma, hemorrhage, and infections. Also can identify changes in hyper-inflammatory conditions for early infection and find out the usefulness of PTX, which is of interest in our research. We used the Jurkat cell which plays the main role of immunity, the human T lymphocytic cell line, commercially purchased for the study of T lymphocytes, to find out the change in immune-paralysis conditions and to find out the effect of PTX. T lymphocytes were co-cultured into stimulated macrophages to find out the effect of hyper-inflammatory macrophages and the effect of PTX injection on T lymphocytes to identify the change of immunity.
THP-1 cells were converted into differential THP-1 cells after 3 days of incubation with PMA injection to develop the propensity of macrophages for the experiment. The study was conducted on an eight-hour basis since TLR4 increased after eight hours of injection of LPS. As a result, TLR4, which is a bacterial infection receptor when injected with LPS, increased when measured with flow cytometry that directly measures the receptor in cells. It was meaningful that TLR4 reduced as PTX capacity increased. The PCR that checks the mRNA expression in gene measurement showed that the mRNA expression of TLR4 increased at LPS injection, but the injection of PTX did not show any change. The PTX reported anti-inflammatory tendencies by mitigating tumor necrosis factor-α (TNF- α) increases, nuclear factor–κB (NF-κB) activation, TLR4 increases, MIF increases, etc. in stimulated cells [5]. As there is no change in the PCR method, it is assumed that it does not affect the gene level. It is estimated the reduction of TLR4 and MIF that react early in the stimulus decrease the phosphorylation of NF-κB indicating a decrease in TNF-α [5–7]. It was conducted using the Jurkat cell, a human acute lymphocytic leukemic cell, which is widely used for research on activation of T lymphocytes that play an important role in immunity. The injection of PGE2, an immune-reducing substance, reduced the number of Jurkat cells by the MTT method. Unlike the restoration of reduced T lymphocyte
by hypertonic saline in our past studies [8], PTX did not restore the reduced T lymphocyte. PTX shows multiple beneficial effects on the inflammatory cascade according to various studies. PTX down-regulates the production of various pro-inflammatory cytokines (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10) which increase in infection, hemorrhage, and shock. Also, inhibit the activation of NF-κB in various cells and inhibits the production of various reactive oxygen species (ROS), including superoxide anion (O2−), hydrogen peroxide (H2O2) which is known to increase during ischemia-reperfusion [7, 9–11]. Despite the many studies on reports of increasing transforming growth factor (TGF)-β and an anti-inflammatory cytokine in coronary artery disease [12, 13], there have been no studies on T lymphocyte. Our research showed no effect on immunity increase by increased T lymphocytes.
The MIF has been shown to not only override the anti-inflammatory effects of glucocorticoid but also to induce TLR4 expression on the surface of the cell, inhibit p53-mediated apoptosis and stimulate proliferation of cells [14]. Therefore, the MIF plays an important role in sepsis by controlling inflammatory reactions, including activations of various cytokines in macrophages, neutrophils, and T lymphocytes [15, 16]. In our study, as with the increase of TLR4 during LPS injection, MIF was also increased to identify the relationship between TLR4 and MIF, and PTX was effective by restoring TLR4 and MIF. As the relationship between MIF and T cell proliferation was also known, the MIF was reduced when the T cell proliferation was reduced by PGE2. This was the same as in our previous study that MIF was increased during the injection of LPS into macrophages, resulting in pro-inflammatory effects, and MIF was decreased in the injection of PGE2 which indicates decreased immune function, along with the reduction of T cell proliferation, to indicate immune-paralysis [8]. In this study, PTX did not restore T cell proliferation and MIF. Moreover, PTX showed a decrease further than PGE2 injection.
The two cells were co-cultured to find out the effects of macrophages on T lymphocytes, which play an important role in the immune system. Generally, the number of lymphocytes in normal healthy people was about 7 to 20 times that of monocyte, but in order to determine the appropriate number of cells for co-culture between two cells in the experiment, the Jurkat cells for MTT measurement evaluation were set at 2 × 106/mL. The differentiated THP-1 cells were co-cultured in various numbers and differentiated THP-1 cells were selected to be 1 × 104/mL by showing similar MTT as shown. The differential THP-1 cell stimulated by LPS was washed with PBS to remove the effects of LPS, placed at the bottom of the culture plate divided into two layers, and the Jurkat cell was placed upstairs to measure the MTT of the Jurkat cells. In the THP-1 cells group stimulated by the LPS, the Jurkat cells decreased compared to the non-stimulated THP-1 cells group showing macrophages in hyper-inflammatory conditions could induce a decrease in T lymphocytes. Banchereau J et al. [1] reported that the Dendritic cell acts as an initiator and modulator for immune response. Munn DH, et al. [2] also reported that macrophage is known to suppress T cell proliferation due to the influence of macrophage colony-stimulating factor (MCSF), and MCSF-derived macrophages were capable of depleting the essential amino acid tryptophan from co-culture. Our study showed similar conclusions. Moreover, our research makes it easier to recognize immunity by measuring the Jurkat cell viability directly. The MTT (T cell proliferation) value of Jurkat cells group injected with PTX and LPS in the macrophages restored the MTT value of Jurkat cells group in the macrophages stimulated only by LPS to the normal level. Besides, as the capacity of the PTX increases, the MTT value of Jurkat cells increased. The IL-2, a cytokine that plays an important role in T cell proliferation, was tested using various methods (RT-PCR, western blots) to check the accuracy of MTT viability assay. The MTT viability assay showed a similar reduction in both groups, however, injection of LPS and PTX restored the control group. In other words, stimulated macrophages reduced T cell proliferation and IL-2. On the other hand, PTX restored the level of the control group [17–20]. Since IL-2 is known to increase immunity by inducing T cell proliferation, the injection of PTX into LPS stimulated macrophages could be determined to increase immunity by increasing T cell proliferation [11].
PTX starts with a substance that improves the microvascular blood flow and is reported to reduce the mortality of neonatal sepsis. Also reduces the activation of NF-κB as well as downregulating CD66b and TNF-α expression as a mechanism to attenuate the various pro-inflammatory neutrophil functions [5]. There was no research on the T cells of PTX before us. In our study, PTX was not able to act directly on the T cells to enhance immunity, but the effect of restoring MIF and TLR4 expression increased in macrophages stimulated by LPS from co-culture. Moreover, PTX restored the reduced MTT value and IL-2 expression to the T cell control group. In conclusion, PTX was found to maintain homeostasis with the restoration of the T cells control group, which is clinically related to IL-2.
There are many limitations. First, studies of intercellular effects and relationships between macrophages and T lymphocytes, excluding studies of intercellular effects, such as neutrophil, should be conducted later. Second, in the co-culture of LPS-stimulated macrophages and T lymphocytes, PTX has restored the T cells proliferation to the level of the control group, but only IL-2 has been conducted in our study, so future research will be needed. However, we understand that MIF as well as IL-2 are involved in T cell proliferation in our previous research, but more research is expected to be needed. Third, for the usefulness of the PTX, further research will be needed, such as animal experiments or clinical trials.