High‐mobility group box‐1 and inter‐alpha inhibitor proteins: In vitro binding and co‐localization in cerebral cortex after hypoxic‐ischemic injury

Abstract

The high‐mobility group box‐1 (HMGB1) protein is a transcription‐regulating protein located in the nucleus. However, it serves as a damage‐associated molecular pattern protein that activates immune cells and stimulates inflammatory cytokines to accentuate neuroinflammation after release from damaged cells. In contrast, Inter‐alpha Inhibitor Proteins (IAIPs) are proteins with immunomodulatory effects including inhibition of pro‐inflammatory cytokines. We have demonstrated that IAIPs exhibit neuroprotective properties in neonatal rats exposed to hypoxic‐ischemic (HI) brain injury. In addition, previous studies have suggested that the light chain of IAIPs, bikunin, may exert its anti‐inflammatory effects by inhibiting HMGB1 in a variety of different injury models in adult subjects. The objectives of the current study were to confirm whether HMGB1 is a target of IAIPs by investigating the potential binding characteristics of HMGB1 and IAIPs in vitro, and co‐localization in vivo in cerebral cortices after exposure to HI injury. Solid‐phase binding assays and surface plasmon resonance (SPR) were used to determine the physical binding characteristics between IAIPs and HMGB1. Cellular localizations of IAIPs‐HMGB1 in neonatal rat cortex were visualized by double labeling with anti‐IAIPs and anti‐HMGB1 antibodies. Solid‐phase binding and SPR demonstrated specific binding between IAIPs and HMGB1 in vitro. Cortical cytoplasmic and nuclear co‐localization of IAIPs and HMGB1 were detected by immunofluorescent staining in control and rats immediately and 3 hours after HI. In conclusion, HMGB1 and IAIPs exhibit direct binding in vitro and co‐localization in vivo in neonatal rats exposed to HI brain injury suggesting HMGB1 could be a target of IAIPs.

Changes in Cellular Localization of Inter-Alpha Inhibitor Proteins after Cerebral Ischemia in the Near-Term Ovine Fetus

“…Similar to our findings in the human brain during development and in neonatal rats, more than 90 percent of cells in the cerebral cortex and white matter of the sheep fetus were positive for IAIPs by immunohistochemistry ( Figure 2 ) [ 18 , 29 ]. These data strongly support the importance of IAIPs for normal brain function and development [ 16 , 17 , 18 , 27 , 29 ].…”

“…Similar to our findings in the human brain during development and in neonatal rats, more than 90 percent of cells in the cerebral cortex and white matter of the sheep fetus were positive for IAIPs by immunohistochemistry ( Figure 2 ) [ 18 , 29 ]. These data strongly support the importance of IAIPs for normal brain function and development [ 16 , 17 , 18 , 27 , 29 ]. Interestingly, in the present study, exposure to I/R was associated with a reduced proportion of cells expressing cytoplasmic IAIPs and a reciprocal increase in the proportion of cells expressing nuclear IAIPs in the cerebral cortex.…”

“…Our previous findings suggested that ischemia resulted in reduced IAIP levels in the CNS of fetal sheep [ 27 ] and that hypoxia was associated with reduced IAIP levels in the cerebral cortex of neonatal rats [ 43 ]. In addition, we have previously found changes in the subcellular localization of IAIPs in neonatal rat brains after exposure to HI brain injury [ 29 ]. However, these studies did not define the specific brain cell types in which the cellular localization was altered by exposure to HI.…”

“…We therefore postulated that endogenous IAIPs in the brain may be affected by HI-related inflammation in the fetal and neonatal brain. We have previously shown changes in the subcellular localizations of IAIPs after HI [ 29 ]. However, in our previous study, we did not examine whether there were differences between types of cells after HI [ 29 ].…”

“…We have previously shown changes in the subcellular localizations of IAIPs after HI [ 29 ]. However, in our previous study, we did not examine whether there were differences between types of cells after HI [ 29 ].…”