Kinetics of Intracellular, Extracellular and Production of Pro-Inflammatory Cytokines in Lipopolysaccharide- Stimulated Human Peripheral Blood Mononuclear Cells

Purpose: To determine the detailed time course of intracellular, extracellular and production of proinflammatory cytokines (TNF-α, IL-1β, IL-1α, IL-6 and IL-8) in normal human peripheral blood mononuclear cells (PBMC). Methods: PBMC were isolated from human whole blood, stimulated by 0.1 μg/ml lipopolysaccharide (LPS) and incubated at 37oC, 5% CO2. Samples were harvested at different time intervals (4, 8, 12, 16, 20 and 24 h) after stimulation. ELISA was employed for the measurement of the extracellular and intracellular cytokine levels of the samples. Results: The release of TNF-α, IL-6 and IL-8 on LPS-stimulated PBMC were significantly higher with concentrations in the range of 3161 ± 162.5 to 4027 ± 361.5 pg/ml (p < 0.001), 3921.5 ± 879.3 to 11628.3 ± 2647.3 pg/ml (p ≤ 0.030), and 4122.0 ± 382.9 to 5898.6±115.8 pg/ml (p ≤ 0.049), respectively compared to intracellular levels that were very low (TNF-α, 23.5 ± 5.0 to 69.5 ± 13.8 pg/ml; IL-6, 22.5 ± 16.5 to 96.5 ± 9.6 pg/ml; and IL-8, 501.1 ± 221.0 to 1452.5 ± 415.7 pg/ml) and remained unchanged during 24 h. In contrast, both IL-1α and IL-1β were secreted gradually. Secretion and production of IL-6 was significantly higher at 8 h (9394.4 ± 846.3 pg/ml; p = 0.002) and at 20 h (11628.3 ± 2647.3 pg/ml; p = 0.014) than other cytokines. Conclusion: The differences in the characteristic kinetics of cytokines may be caused by different mechanisms of secretion and function. IL-1, TNF-α and IL-8 play a role as proinflammatory cytokines, whereas IL-6 consecutively plays a dual role as pro-inflammatory cytokine and anti-inflammatory.


INTRODUCTION
Cytokines are low molecular weight proteins produced by cells in response to a variety of stimuli. After genesis, they influence the behavior of target cells [1,2]. These molecules are essentially involved in a number of biological processes, including cell proliferation, inflammation, immunity, migration, fibrosis, repair and angiogenesis [3]. Cytokines that promote inflammation are known as pro-inflammatory cytokines [4]. Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), Interleukin-1α (IL-1α), Interleukin-6 (IL-6) and Interleukin-8 (IL-8) are pro-inflammatory cytokines which even though are dissimilar proteins transcribed from different genes, share important roles in acute inflammation [5].
TNF-α is involved in phagocyte activation, endotoxic shock, tumor cytotoxicity and IL-1 plays important roles in cell proliferation and differentiation and pyrexia. Meanwhile, IL-6 has a role in cell differentiation into plasma cells, immunoglobulin production and IL-8 recruits and retains neutrophils at sites of inflammation [6].
Many studies on the kinetics of cytokine production have been carried out in experimental animals, whole blood models and isolated cells [7][8][9][10][11][12]. Kwak et al reported intracellular and extracellular cytokine production by human mixed mononuclear cells in response to group B Streptococci and E. coli LPS [13]. However the effect of Salmonella enteritica LPS on the kinetics of intracellular, extracellular and production of these pro-inflammatory cytokines in normal human PBMC not been reported. TNF-α is one of the prominent pro-inflammatory cytokines in cancer and rheumatoid arthritis and some studies have reported that TNF has a key role in the regulation of inflammatory processes [7,8,14,15].
However, the roles of other pro-inflammatory cytokines are not well studied. It is interesting to evaluate the involvement of other proinflammatory cytokines in inflammation induced by lipopolysaccharide (LPS), especially IL-6 which has been reported to possess both proand anti-inflammatory properties. In the present study, the detailed time course of intracellular, extracellular and production of these proinflammatory cytokines was determined in normal human peripheral blood mononuclear cells (PBMC) induced by Salmonella enteritica LPS.

Isolation of PBMC
Human whole blood from healthy volunteers (≥ 18 years old) was collected for the isolation of PBMC. The inclusion criteria were considered: non-smoker, fasted overnight and did not take any medicine or supplements. Venous blood was collected in heparinized tubes and processed immediately.

Kinetic study
PBMC were stimulated with 0.1 μg/ml of LPS from Salmonella enteritica (Sigma, Steinheim, Germany) and incubated at 37ºC, 5% CO 2 . Samples were harvested at different time intervals (4,8,12,16,20 and 24 h) after stimulation with LPS. Supernatants were collected for measuring extracellular cytokines. Pelleted cells were washed with RPMI-1640 and lysed based on the procedure described by Hazuda et al [10] which used lysis buffer (40mM; 50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 1 mM dithiothreitol) plus 2 mM PMSF followed by sonication for 30 s, with modification by changing lysis buffer with water followed by gentle shaking for 10 s and left at room temperature for 1 h for measuring intracellular cytokines. This modification was performed to avoid the use of chemicals which may influence the stability of the cytokines. A control group was also performed to examine cytokine release but in the absence of added LPS. The cells used as a control group and stimulated group using LPS were taken from the same subjects. Cytokine production was determined by measuring the intracellular and extracellular levels of cytokines.

Statistical analysis
All statistical analyses were performed using SPSS® Statistic 20. One-way ANOVA, followed by a post-test (Tukey's multiple comparison) were used when significant differences at p<0.05 were present. Cytokine assays by ELISA were determined in duplicate, and data were obtained from three different donors. The results are expressed as mean ± standard error of the mean (n = 3).

RESULTS
The results show that the levels of TNF-α secreted in response to LPS stimulation were significantly higher (3161 ± 162.5 to 4027 ± 361.5 pg/ml; p<0.001) than those of intracellular TNF-α and the control group throughout the 24 h incubation period. Moreover, the results showed no significant difference between intracellular and extracellular TNF-α levels in control group. Additionally, there was no significant difference between LPS-stimulated and non-stimulated intracellular TNF-α levels ( Figure 1A). # p < 0.05, ## p < 0.01, ### p < 0.001 for the LPS-stimulated extracellular cytokines versus the intracellular. *p < 0.05, **p < 0.01, ***p < 0.001 for the LPS-stimulated extracellular cytokines versus the non-stimulated. + p < 0.05, ++ p < 0.01, +++ p < 0.001 for the non-stimulated extracellular cytokines versus the intracellular. =p < 0.05, ==p < 0.01 for the LPS-stimulated intracellular cytokines versus the non-stimulated In response to LPS stimulation, the intracellular IL-1α levels were significantly higher at 4 -8 h (843.0 ± 101.1 -1051 ± 29.0 pg/ml; p≤0.002) than the extracellular and control groups but no significant difference was observed at 12 -24 h. Additionally, extracellular IL-1α concentrations in response to LPS-stimulation were significantly higher compared to non-stimulated extracellular at 8 h (586.2 ± 91.8 pg/ml; p = 0.003) and 20 -24 h (982.5 ± 176.2 to 1013.2 ± 171.7 pg/ml; p≤0.013). The results also showed that there was no significant difference between extracellular and intracellular levels of IL-1α in non-stimulated cells ( Figure 1B).
The levels of IL-6 released in response to LPS stimulation were significantly higher than its intracellular levels and the control group throughout 4-24 h (3921.5 ± 879.3 to 11628.3 ± 2647.3 pg/ml; p≤0.030). Moreover, the results showed that there was no significant difference between non-stimulated IL-6 extracellular and intracellular and between stimulated and non-stimulated IL-6 intracellular ( Figure 1D). Figure  1E shows that the levels of IL-8 secreted increased gradually in response to LPS and was significantly higher than its intracellular levels and the control group throughout 4-24 h (4122.0 ± 382.9 to 5898.6 ± 115.8 pg/ml; p≤0.049). The results also showed that the concentrations of non-stimulated IL-8 extracellular increased gradually and were significantly higher than its intracellular levels throughout 8 -24 h (3209.3 ± 291.4 to 4481.6 ± 420.9 pg/ml; p = 0.001). Figure 2A shows that in the non-stimulated PBMC, IL-8 was the most concentrated cytokine as it was released gradually and reached its maximum level at 4481 ± 420.9 pg/ml at 24 h. This was followed by IL-6 which reached its maximum level of 2732.4±340.3 pg/ml at 20 h. Whereas the levels of TNF-α, IL-1α and IL-1β were very low and remained unchanged during 24 h time course. Figure 2B shows that in LPSstimulated cells, the extracellular levels of IL-6 were significantly higher at 8 h (9394.4 ± 846.3; p=0.002) and 20 h (11628.3 ± 2647.3pg/ml, p=0.014) as compared to other cytokines, followed by IL-8, TNF-α, IL-1β and IL-1α in order of decreasing concentrations. In addition, there was no significant difference between IL-1α and IL-1β secretion during the 24 h period.
In the intracellular non-stimulated PBMC, the levels of IL-1β gradually increased until it reached maximum value at 16 h (1306.5 ± 491.0 pg/ml) and then decreased onwards. While the levels of IL-1α increased gradually until it reached a maximum value at 20 h (741.6 ± 79.1 pg/ml) and then decreased. Whereas TNF-α, IL-6 and IL-8 were in low levels (≤159.6 ± 17.0 pg/ml) and remained unchanged during the 24 h period ( Figure 3A).  Figure 4 shows the time course of proinflammatory cytokine production in that IL-6 was the highest cytokine produced at 8 to 24 h.

DISCUSSION
The results indicated that there were differences in the kinetics of intracellular, extracellular and production of cytokines in human PBMC stimulated by LPS. TNF-α, IL-6 and IL-8, were secreted directly in high quantities, but the intracellular level of these cytokines were very low and remained unchanged during 24 h of incubation. On the contrary, both IL-1α and IL-β were secreted gradually. The difference in their characteristics was due to the different functions and different pathways of cytokine secretion. In the macrophages, once LPS stimulates via tolllike receptor 4 (TLR4), the type II transmembrane precursor pro-TNF is quickly synthesized and builds up in the Golgi and delivered to the cell surface [17][18]. Then it is cleaved by TNF alpha converting enzyme (TACE) and A disintegrin and a metalloproteinase 17 (ADAM17) [19].
Likewise, LPS stimulation leads to the accumulation of newly synthesized IL-6 in the Golgi complex in macrophages, which is subsequently loaded into tubo-vesicular carriers that bud off the Trans-Golgi network (TGN) either as the sole labeled content or jointly with TNF [20]. Since IL-6 is a soluble cytokine rather than a trans-membrane cytokine like TNF, it uses multiple carriers (including p230 and Golgin-97) for its export from the Golgi [21]. After export from the Golgi, both TNF and IL-6 colocalize with recycling endosomes (REs) in macrophages during their transport to the cell surface following LPS stimulation [18]. Furthermore, it was shown that IL-6 and TNF were localized in different subcompartments of REs [20]. It was suggested that REs may in addition function to sort these two cytokines for release at different points on the cell surface. Additionally, TNF-α and IL-6 are mainly secreted in the supernatant because they have a hydrophobic group in their precursor amino acid sequences that are most likely signal peptides [7].
In contrast, IL-1 is released by a non-classical secretory route in which a number of cytokines synthesized in the cytoplasm are released through the plasma membrane without passing through the traditional organelles such as REs and Golgi complex [22]. IL-1β lacks a signal peptide and is produced on free ribosomes in the cytoplasm and cleaved by caspase-1 of the inflammasome preceding its release [23][24]. How these cytokines break out from cells is still mainly a mystery. Numerous pathways and mediators have been called upon, including ATPbinding cassette (ABC) transporters, secretory lysosomes, endosomes, membrane blebbing [25][26][27] and most newly, transport via multi-vesicular body-derived exosomes [28]. Although there are functional similarities between IL-1α and IL-1β [29], yet they are different in terms of secretion. The phosphorylation of the intracellular precursor of IL-1α may facilitate the processing and release of IL-1α, but this phosphorylation does not take place on IL-1β [11]. The mechanism of how IL-8 secreted is still poorly understood.
The presence of extracellular and intracellular cytokines, respectively, even without stimulation by LPS may be due to the highly sensitive PBMC in response to media or culture conditions [30][31][32][33][34].
These differences in the kinetics of cytokine secretion and production undoubtedly provide a reflection of the functional differences of these mediators. Although TNF and IL-1 share numerous biological activities, the major distinction being that TNF has no direct effect on lymphocyte proliferation. Although IL-6 shares several activities with IL-1, including induction of pyrexia and the production of acute phase proteins, IL-6 is considered the most important hepatocyte-inducer for synthesis of acute phase proteins. In contrast to these pro-inflammatory effects, IL-6 mediates several anti-inflammatory effects by terminating up-regulatory inflammatory cascade and inhibits IL-1 and TNF synthesis [35]. On the other hand, IL-8 mediates the recruitment and activation of neutrophils in inflamed tissue [4][5].
On extracellular LPS-nonstimulated, the level of IL-8 was significantly higher compared to other cytokines. The level of IL-8 gradually increased until it reached a maximum value at 20 hours, while other cytokines remained unchanged during 24 h. This finding can be explained by the fact that IL-8 is an autocrine regulator of its self production in monocytes [36]. However, extracellular LPS-stimulated PBMC showed that the level of IL-6 was higher than those of other cytokines. This profile may be explained by the fact that many cytokines such as TNF-α, IL-1 induce the release of IL-6 [5]. It was also showed that at 20 h the level of IL-6 increased but at the same time the level of IL-1β and TNF-α decreased, which may be caused by the antiinflammatory effects of IL-6 by suppressing IL-1β and TNF-α at the level of 11628 pg/ml. This finding is in agreement with those reported by Schindler et al. [7]. IL-6, although mostly regarded as a pro-inflammatory cytokine, also has many regenerative and anti-inflammatory activities. Pro-inflammatory responses of IL-6 are mediated by trans-signaling. IL-6 will bind to soluble interleukin-6 receptor (sIL-6R), afterwards this IL-6/sIL-6R complex binds to glycoprotein 130 (gp130) receptors to produce pro-inflammatory signal. Whereas the antiinflammatory response of IL-6 is mediated by classic signaling. In the classic signaling IL-6 first binds to the membrane-bound non signaling receptors IL-6R (mbIL-6R) then this complex binds to two molecules of gp130 and leads to IL-6-signal transduction [37].
However there has been no explanation of how these two opposing activities can occur. That interesting phenomenon can be explained by our findings. The results show that when LPS triggers the production of pro-inflammatory cytokines, IL-6 led excretion of other cytokines, this was shown by its highest extracellular concentration compared with other cytokines at 24 h incubation. IL-6 level was gradually increased to maximum level during 20h and at that period pro-inflammatory effect was produced. When the maximum level (11628.3 ± 2647.3 pg/ml) was reached, sIL-6R were saturated (at this time IL-6 release rate was higher than Fas ligation, resulting in ADAM17 mediated shedding of membrane IL-6R (mIL-6R) from the surface), that is when IL-6 start to bind mIL-6R, classic signaling will occur and produces anti-inflammatory activity. This process may have been caused by the affinity of IL-6 to the sIL-6R being higher than mbIL-6R.
Anti-inflammatory activity also occurs when IL-6 level reached the maximum level and some of IL-6 start binding to mbIL-6R, this binding causes a decline of extracellular IL-6 after 20 h incubation. It may be that at the same time, IL-6 starts to inhibit secretion of TNF-α and IL-1β. This explanation is also supported by Figure 4 which shows that IL-6 production was highest from 8 h until 24 h incubation. The fact that IL-6 suppresses IL-1β and TNF production was reported by Schindler et al [7] and also stated by Kwak et al [13]. Furthermore, IL-6 also stimulates synthesis of IL-1 receptor antagonist (IL-1ra) [35].

CONCLUSION
The differences in the characteristic kinetics of cytokines may be caused by different mechanisms of secretion and function. IL-1, TNF-α and IL-8 are proinflammatory cytokines, whereas IL-6 acts as a pro-inflammatory cytokine or an anti-inflammatory cytokine. The change in its activity may be affected by its level.