Effect of inter-alpha inhibitor proteins (IAIP) on neutrophil functions

[Speaker] Soe Soe Htwe:1
[Co-author] Hidenori Wake:1, Keyue Liu:1, Kiyoshi Teshigawa:1, Barbara S Stonestreet:3, Yow Pin Lim:2, Masahiro Nishibori:1
1:Pharmacology Department, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 3rd year doctoral course student, Japan, 2:Department of Pathology and Laboratory Medicine, Warren Alpert Medical School of Brown University and Pro Thera Biologics, Inc. , USA, 3:Department of Pediatrics, Women & Infants Hospital of Rhode Island, The Alpert Medical School of Brown University, USA

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. As a part of the innate immune system, neutrophils are activated and produce the pro-inflammatory mediators and reactive oxygen species (ROS) and release granular enzymes. Administration of protease inhibitors have been proposed as a therapeutic strategy to restore the balance between proteases and protease inhibitors in sepsis. One such inhibitor is the inter-alpha inhibitor proteins (IAIP). It is a serine protease inhibitor found at relatively high concentrations in plasma (400-800 mg/L). It is composed of heavy and light chain polypeptide subunits that are covalently linked by a glycosaminoglycan chain. The light chain, bikunin, contains the serine protease inhibitory activity and it has been widely used as a drug for patients with inflammatory disorders. The purpose of the present study was to examine the effects of IAIP on neutrophil functions, especially focused on their morphology, capillary passage, adhesion on endothelial monolayer and ROS production.
Purified human neutrophils were treated with IAIP or bikunin and morphological changes were observed under In Cell Analyzer 2000. For detecting the passage of neutrophils through the microvasculatures, Micro Channel Array Flow Analyzer (MC-FAN) was used. Neutrophil adhesions on endothelial monolayer (EA.hy926) was evaluated by measuring the residual cells after removing the non-adherent cells. Neutrophil's extracellular ROS production was measured by chemiluminescene intensity using Flexstation 3.

We demonstrated that IAIP maintains neutrophil's spherical shape with smooth cellular surface. But bikunin (light chain of IAIP) had no effect on neutrophil morphological changes compared with IAIP. This IAIP-treated with spherical shape neutrophils can easily pass through the microcapillary slit and were prevented from the entrapment inside the slits. Co-incubation of human blood neutrophils with confluent inactivated human vascular endothelial monolayer shows that the neutrophil adhesion on endothelial monolayer decreased in IAIP-treated group. IAIP concentration-dependently inhibited the spontaneous extracellular ROS production.
All these results suggested that IAIP-induced morphological changes render neutrophils quiescent with respect to the passage through microvasculature, cell adhesions to vascular endothelial cells and spontaneous release of reactive oxygen species (ROS).

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