Identification and modulation of the allograft inflammatory factor-1 (AIF-1) homologous in the medicinal leech Hirudo Medicinalis.

Allograft inflammatory factor-1 (AIF-1) is a 17 kDa cytokine-inducible calcium-binding protein that in vertebrates plays an important role in allografts immune response and its expression is mostly limited to the monocyte/macrophage lineage. Recently it was assumed that AIF-1 was a novel molecule involved in inflammatory responses. To better clarify this aspect in the present study we investigated the expression of AIF-1 after bacterial challenge, wounds and tissue transplants and its potential role in regulating the innate immune response in an invertebrate model, the medicinal leech (Hirudo medicinalis). The analysis of an EST library from H. medicinalis CNS, revealed the presence of a gene, named Hmaif-1/alias Hmiba1, showing a high homology with vertebrate aif-1. Immunohistochemistry using an anti-HmAIF-1 polyclonal antibody showed that this protein is constitutively present in spread, CD68+ macrophage-like cells. A few hours after pathogen bacterial injection in the body wall, the amount of these immunopositive cells increases at the injected site, co-expressing HmAIF-1 and the common leukocyte marker CD45. A similar overview was observed in the early stages of wound healing, especially at 24 hours after injury, with a lot of immunopositive cells around the lesion site. Moreover here we demonstrated that the recombinant protein HmAIF-1 induced a massive angiogenesis and it was also a potent chemoattractant for macrophages. After rHmAIF-1 stimulation, macrophage-like cells co-expressed the macrophage marker CD68 and the surface glycoprotein CD45, which in Vertebrates is implicated in the integrinmediated adhesion of macrophages and plays a key role in regulating the functional responsiveness of cells to chemoattractants. We therefore hypothesized that CD45 could play a role for leech macrophage-like cells activation and migration towards the inflammation site and we examined its potential effect on HmAIF-1-induced signaling.