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.