DISCUSSION
A large number of studies have shown that leukocytes play an important role in inducing human inflammatory diseases (Peiseler, Kubes, 2019; Suzuki, 2017; Powell, Huttenlocher, 2016; Jasper, McIver, Sapey, Walton, 2019; Kovtun, Messerer, Scharffetter-Kochanek, Huber-Lang, Ignatius 2018; Williams, Chambers, 2016; Wright, Moots, Bucknall, Edwards, 2010; Suzuki, 2018; Zhang, 2019; Mortaz, Alipoor, Adcock, Mumby, Koenderman, 2018). The migration of leukocytes into inflamed tissues behaves like a double-edged sword, not only help to remove invasive microorganisms and other foreign entities, but also contribute significantly to the pathophysiology of inflammatory diseases. In this study, the effect of intestinal alkaline phosphatase on TNF-α and IL-6 production by freshly extracted human leukocytes was successfully investigated in the presence and absence of endotoxin LPS using our cell-based model (Figure 2 and 3). LPS stimulates human leukocytes (mainly human neutrophils) and increases the production of TNF-α and IL-6. We demonstrated that IAP can effectively inactivate LPS at neutral pH 7.5 (Table 1). Interestingly, IAP inhibited leukocytic TNF-α secretion to a similar extent regardless of whether LPS and recIAP were added simultaneously to leukocytes or LPS was incubated with recIAP in advance before being added to leukocytes. This result suggests that IAP can inhibit TNF-α secretion not only by first inactivating the effect of LPS when the endotoxin is present, but also by directly acting upon leukocytes. Therefore, our finding that the IAP inhibits TNF-α and IL-6 secretion in freshly extracted human leukocytes in presence and absence of LPS indicates a therapeutic potential of the IAP for the treatment of diseases related to dysregulated production of TNF-α /IL-6 in leukocytes. In other words, the IAP administration (Peters, 2016a) can be used to treat not only LPS-related diseases such as sepsis-related, renal injury, but also diseases related to upregulation of TNF-α /IL-6. Our key finding greatly extends the application of the IAP as therapeutics to a much broader therapeutic space and disease landscape (Poelstra, Bakker, Klok , Hardonk, Meijer, 1997; Peters, 2016a; Lukas,2010; Peters, 2016b). In addition, our freshly extracted leukocyte-based assay is a promising quality control bioactivity assay for the commercialization of IAP injectable drugs (Kaliannan, 2013; Peters, 2016a; Mortaz, Alipoor, Adcock, Mumby, Koenderman, 2018; Peters, 2016b; Kiffer-Moreira, 2014; Qian, 2010).
AM-Pharma is currently manufacturing and investigating recIAP injections in clinical trials that are aimed to treat LPS-related diseases, such as sepsis-related renal injury and colitis (Peters, 2016a). It was reported that AM-Pharma’s recIAP injection has a safe injection range for humans between 250, 500, and 1000U kg-1(Peters, 2016a). If it is assumed that an average person’s body weight is 70 kg, each treatment approximately requires a dose of 12,500, 25,000, or 50,000 U (Peters, 2016a). In this study, the effective dose of recIAP that inhibits TNF-α and IL-6 was 2.5-5U ml-1, which when scaled up to a person with an average weight of 70kg (5L blood, 3L plasma) translates into 7,500- 15,000U for each treatment. For reference, AM-Pharma’s recIAP has a concentration of 7036U (11.26mg ml-1 per injection) (Peters, 2016b) and has a specific activity of 625U/mg and purity of 99%. The specific activity of recIAP used in this study is 578 U mg-1, which is close to that of AM-Pharma’s recIAP injection. Thus, the recIAP used in our study proves to be cost-effective within the range of known human-safe dosing(Peters, 2016a; Peters, 2016b).
In addition to inhibiting LPS activity (Figures 3-A and B) (Poelstra, Bakker, Klok, Hardonk, Meijer, 1997), IAP might also act on TNF-α secretion through ATP dephosphorylation (Peters, 2018; Peters,2015). IAP dephosphorylates its substrate ATP and generates ADP, AMP and adenosine. This study found that in the absence of LPS, IAP inhibits TNF-α and IL-6 secretion through dephosphorylating ATP, ADP, AMP in order to generate adenosine. Our experiments demonstrated that adenosine and AMP at least partially inhibit human leukocytic secretion of TNF-α (Figure 6C and D) (Trautmann, 2009). However, it is possible that intestinal alkaline phosphatase also dephosphorylates other substrates, such as degraded cellular substances GTP, CTP, TTP, UTP, other nucleic acids, etc. For example, the rapid degradation of RNA can generate an ample source of dephosphorylation targets for IAP. Moreover, this study cannot exclude the possibility that particular cell surface receptors or signaling pathways which utilize phosphorylation are specifically targeted by alkaline phosphatase and subsequently affect the production of TNF-ɑ and IL-6 by human leukocytes(Labugger, Organ, Collier, Atar, Van Eyk, 2000).
In conclusion,we found that IAP can inhibit TNF-α and IL-6 secreted by freshly extracted human leukocytes in the absence of endotoxin LPS. IAP is a promising injectable anti-inflammatory drug candidate for treatment of human diseases with dysregulated human leukocyte infiltration and TNF-α/ IL-6 production. We further found that intestinal alkaline phosphatase inhibits leukocyte TNF-α and IL-6 secretion through dephosphorylating ATP, ADP, AMP, and other substrates besides LPS. The leukocyte-based cellular model developed in this study can act as a promising bioactivity assay for quality control to be used for the commercialization of an injectable IAP drug.