Homolog of allograft inflammatory factor-1 induces macrophage migration during innate immune response in leech

Abstract

Allograft inflammatory factor-1 (AIF-1) is a 17-kDa cytokine-inducible calcium-binding protein that, in vertebrates, plays an important role in the allograft immune response. Its expression is mostly limited to the monocyte/macrophage lineage. Until recently, AIF-1 was assumed to be a novel molecule involved in inflammatory responses. To clarify this aspect, we have investigated the expression of AIF-1 after bacterial challenge and its potential role in regulating the innate immune response in an invertebrate model, the medicinal leech (Hirudo medicinalis). Analysis of an expressed sequence tag library from the central nervous system of Hirudo revealed the presence of the gene Hmaif-1/alias Hmiba1, showing high homology with vertebrate aif-1. Immunohistochemistry with an anti-HmAIF-1 polyclonal antibody revealed the constitutive presence of this protein in spread CD68(+) macrophage-like cells. A few hours after pathogen (bacterial) injection into the body wall, the amount of these immunopositive cells co-expressing HmAIF-1 and the common leucocyte marker CD45 increased at the injected site. Moreover, the recombinant protein HmAIF-1 induced massive angiogenesis and was a potent chemoattractant for macrophages. Following rHmAIF-1 stimulation, macrophage-like cells co-expressed the macrophage marker CD68 and the surface glycoprotein CD45, which, in vertebrates, seems to have a role in the integrin-mediated adhesion of macrophages and in the regulation of the functional responsiveness of cells to chemoattractants. CD45 is therefore probably involved in leech macrophage-like cell activation and migration towards an inflammation site. We have also examined its potential effect on HmAIF-1-induced signalling.

AIF-1 and RNASET2 Play Complementary Roles in the Innate Immune Response of Medicinal Leech

“…As previously demonstrated [2, 3], double immunofluorescence assays confirmed that the two proteins are constitutively expressed in unlesioned (Fig. 5a) and in PBS-injected animals (Fig.…”

“…We have previously reported that AIF-1 and RNASET2 are significantly upregulated at 6 and 24 h following LPS injection, and that this change in their expression is involved in leech macrophage recruitment [2, 3]. However, the underlying mechanisms by which these two factors operate are still poorly characterized.…”

“…In medicinal leeches, LPS injection is known to induce a massive migration and activation of immunocompetent cells, which are mainly localized in the connective tissue and near the body wall epithelium [2, 3]. Double immunofluorescence experiments of LPS-injected leeches showed a massive recruitment of both CD11b + /RNASET2 + granulocytes and AIF-1 + /RNASET2 + macrophages underneath the epithelium and near the muscular fibers, with a signal specificity for each antibody in keeping with the data obtained by the previous Western blot analysis.…”

“…Moreover, the AIF-1 protein actively secreted by LPS-activated, resident macrophages contributes to immune cell recruitment as well [3, 4]. At later stages (6 h after treatment), a second boost of RNASET2 expression ensues to maintain the inflammatory state by means of recruitment of further macrophages (as shown by the resumed AIF-1 expression detected by Western blot analysis at 24 h) in order to clean the infected area from bacterial debris.…”

“…In fact, our previous investigations showed that injection in leeches of human cytokines and growth factors promotes hematopoiesis, vascular growth, immune cell migration, and myofibroblast differentiation [17]. Among these factors, AIF-1 and RNASET2 have recently been shown to play a pivotal role in the leech immune response and tissue repair [2, 3, 11]. Indeed, expression of both factors is significantly increased following lipopolysaccharides (LPS) or bacterial infection in leeches.…”