Allograft Inflammatory Factor-1 Protein Research Articles

Allograft Inflammatory Factor-1 in Metazoans: Focus on Invertebrates.

Simple SummaryDuring their life, all living organisms defend themselves from pathogens using complex strategies. Vertebrates and invertebrates share mechanisms and molecules that guarantee their overall bodily integrity. Allograft inflammatory factor-1 (AIF-1) is a protein extensively studied in vertebrates, and especially in mammals. This factor, generally involved in inflammation events occurring upon pathogenic infection or tissue injury, is linked to several important human diseases. This review collects data on the presence and role of AIF-1 in invertebrates, which are still poorly investigated organisms. Multiple alignment and phylogenetic analysis reveal that AIF-1 is conserved in vertebrates and invertebrates, suggesting similarity of functions. In some invertebrate species, the expression of AIF-1 increases considerably after a bacterial challenge, indicating that it plays a key role during the immune responses. This review highlights the importance of studying this protein in invertebrates as a way to improve our knowledge of innate immunity mechanisms and to better understand inflammatory regulation events in mammals.Allograft inflammatory factor-1 (AIF-1) is a calcium-binding scaffold/adaptor protein often associated with inflammatory diseases. Originally cloned from active macrophages in humans and rats, this gene has also been identified in other vertebrates and in several invertebrate species. Among metazoans, AIF-1 protein sequences remain relatively highly conserved. Generally, the highest expression levels of AIF-1 are observed in immunocytes, suggesting that it plays a key role in immunity. In mammals, the expression of AIF-1 has been reported in different cell types such as activated macrophages, microglial cells, and dendritic cells. Its main immunomodulatory role during the inflammatory response has been highlighted. Among invertebrates, AIF-1 is involved in innate immunity, being in many cases upregulated in response to biotic and physical challenges. AIF-1 transcripts result ubiquitously expressed in all examined tissues from invertebrates, suggesting its participation in a variety of biological processes, but its role remains largely unknown. This review aims to present current knowledge on the role and modulation of AIF-1 and to highlight its function along the evolutionary scale.

AIF1 was identified as an up-regulated gene contributing to CSFV Shimen infection in porcine alveolar macrophage 3D4/21 cells.

Classical swine fever (CSF) is a disease that is characterized by diffuse hemorrhaging, high fever, and high mortality rates. The pro-inflammatory characteristics of allograft inflammatory factor 1 (AIF1) have been well documented; however, insufficient attention has been given to porcine AIF1. In the present study, AIF1 was identified as a key player contributing to CSFV Shimen infection in porcine alveolar macrophage (PAM) 3D4/21 cell line. Our evaluation showed that AIF1 mRNA and protein are expressed at a time-dependent high level in CSFV Shimen-infected PAM 3D4/21 cells. The transcription and translation of IL6 were also significantly upregulated in infected PAM 3D4/21 cells. By utilizing overexpression RNAs approach, we showed that the cellular AIF1 induced an increased IL6 in PAM 3D4/21 cells. Furthermore, silencing of AIF1 suppressed CSFV Shimen-induced IL6 production in PAM 3D4/21 cells and also inhibited CSFV replication, whereas overexpression of recombinant AIF1 was beneficial for the replication of CSFV Shimen and promoting IL6 production in CSFV Shimen-infected PAM 3D4/21 cells. It is suggested CSFV Shimen induced IL6 in PAM 3D4/21 cells via AIF1 activation, which help clarify the AIF1-related inflammatory processes that occur on CSFV Shimen infected macrophages.

Allograft Inflammatory Factor-1 Mediates Macrophage-Induced Impairment of Insulin Signaling in Adipocytes

Background/Aims: Allograft inflammatory factor-1 (AIF-1) is an inflammatory cytokine produced mainly by macrophages within human white adipose tissue. Its expression is increased in obese subjects and positively correlated with insulin resistance. The purpose of this study is to characterize the regulatory role of AIF-1 in insulin signaling of adipocyte. Methods: AIF-1 was over-expressed via transfection of AIF-1 cDNA into murine RAW 264.7 macrophages, and the constitutive expression of AIF-1 was decreased via transfection of targeting siRNA. Murine 3T3L1 adipocytes were treated with macrophage-conditioned medium or AIF-1 protein. Intracellular lipid accumulation was assayed by oil red O stain. Reactive oxygen species production was determinated by a flow cytometer and adipokine secretion was measured with ELISA. Glucose uptake was detected using the glucose oxidase method and insulin-signal-transduction related molecules were analyzed by Western blot. Results: Short term (48 h) AIF-1 treatment slightly promoted intracellular lipid storage in differentiating 3T3L1 cells. The protein stimulated reactive oxygen species production, provoked TNFα, IL6, resistin, but suppressed adiponectin release and insulin-stimulated glucose uptake both under normal basal and insulin resistance conditions. Furthermore, AIF-1 induced NF-κB activation, inhibited PPARγ expression, GLUT4 translocation to plasma membrane and Akt phosphorylation. Conclusion: Macrophage-derived AIF-1 up-regulated reactive oxygen species production, adipokine TNFα, IL6, resistin release, and inhibited adiponectin secretion. Moreover, it suppressed insulin-stimulated glucose uptake by down-regulating insulin signaling. Thus, AIF-1 could be related to obesity-related diseases.

Molecular and expression analysis of the Allograft inflammatory factor 1 (AIF-1) in the coelomocytes of the common sea urchin Paracentrotus lividus

Allograft inflammatory factor 1 (AIF-1) is a highly conserved gene involved in inflammation, cloned and characterized in several evolutionary distant animal species. Here, we report the molecular identification, characterization and expression of AIF-1 from the common sea urchin Paracentrotus lividus. In this species, AIF-1 encodes a predicted 151 amino acid protein with high similarity to vertebrate AIF-1 proteins. Immunocytochemical analyses on coelomocytes reveal localization of the AIF-1 protein in amoebocytes (perinuclear cytoplasmic zone) and red sphaerulocytes (inside granules), but not in vibratile cells and colorless sphaerula cells. The significant increase of AIF-1 expression (mRNA and protein) found in the coelomocytes of the sea urchin after Gram + bacterial challenge suggests the involvement of AIF-1 in the inflammatory response. Our analysis on P. lividus AIF-1 contributes to elucidate AIF-1 function along the evolutionary scale and consolidate the key evolutionary position of echinoderms throughout metazoans with respect to the common immune paths.

Allograft Inflammatory Factor-1 Links T-Cell Activation, Interferon Response, and Macrophage Activation in Chronic Kawasaki Disease Arteritis.

Kawasaki disease (KD) is widely viewed as an acute arteritis. However, our pathologic studies show that chronic coronary arteritis can persist long after disease onset and is closely linked with arterial stenosis. Transcriptome profiling of acute KD arteritis tissues revealed upregulation of T lymphocyte, type I interferon, and allograft inflammatory factor-1 (AIF1) genes. We determined whether these immune responses persist in chronic KD arteritis, and we investigated the role of AIF1 in these responses. Gene expression in chronic KD and childhood control arteries was determined by real-time reverse-transcriptase polymerase chain reaction, and arterial protein expression was determined by immunohistochemistry and immunofluorescence. Allograft inflammatory factor-1 small-interfering ribonucleic acid macrophage treatment was performed to investigate the role of AIF1 in macrophage and T lymphocyte activation. Allograft inflammatory factor-1 protein was highly expressed in stenotic KD arteries and colocalized with the macrophage marker CD68. T lymphocyte and interferon pathway genes were significantly upregulated in chronic KD coronary artery tissues. Alpha interferon-induced macrophage expression of CD80 and major histocompatibility complex class II was dependent on AIF1, and macrophage expression of AIF1 was required for antigen-specific T lymphocyte activation. Allograft inflammatory factor-1, originally identified in posttransplant arterial stenosis, is markedly upregulated in KD stenotic arterial tissues. T lymphocyte and type I interferon responses persist in chronic KD arteritis. Allograft inflammatory factor-1 may play multiple roles linking type I interferon response, macrophage activation, and antigen-specific T lymphocyte activation. These results suggest the likely importance of lymphocyte-myeloid cell cross-talk in the pathogenesis of KD arteritis and can inform selection of new immunotherapies for clinical trials in high-risk KD children.

Allograft inflammatory factor-1 is an independent prognostic indicator that regulates β-catenin in gastric cancer

Previous studies have revealed that expression of allograft inflammatory factor-1 (AIF-1) protein appears to be increased in malignancies and is correlated with a poorer prognosis in cervical cancer, while its role in gastric cancer has not been reported. We analyzed the expression of AIF-1 in 78 cancer lesions and the corresponding non-cancerous tissues by immunohistochemistry. In contrast with other cancers, we found that AIF-1 protein levels were significantly decreased in 53 of the 78 (67.9%) gastric cancer tissues when compared with the matched normal tissues. This was further confirmed using 7 pairs of fresh gastric cancer tissues and matched adjacent normal tissues. Low tumoral AIF-1 expression was significantly correlated with less favorable clinicopathological characteristics, as well as with reduced overall survival (P<0.001) in the gastric cancer patients. Furthermore, knockdown of AIF-1 obviously increased proliferation, migration and β-catenin expression in BGC-823 and SGC-7901 gastric cancer cells. Taken together, for the first time, we provide evidence that the level of AIF-1 expression may serve as a protective prognostic indicator for gastric cancer.

Expression of allograft inflammatory factor-1 (AIF-1) in response to bacterial challenge and tissue injury in the pearl oyster, Pinctada martensii

Allograft inflammatory factor-1 (AIF-1), an interferon (IFN)-γ-inducible calcium-binding cytokine, is associated with the inflammatory response and defense. We cloned and analyzed the expression pattern of the AIF-1 gene of the pearl oyster Pinctada martensii, hereafter designated PmAIF-1. The full-length PmAIF-1 cDNA is 946bp in length and consists of a 5′-untranslated region (UTR) of 120bp, a 3′-UTR of 376bp, and an open reading frame (ORF) of 450bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 17kDa. Sequence analysis reveals that PmAIF-1 contains two EF hand Ca+2-binding motifs like those in previously characterized AIF-1s while alignment with known AIF-1 protein sequences reveals higher similarity to invertebrate orthologs than to those of vertebrates.Quantitative PCR analysis reveals that PmAIF-1 is constitutively expressed, with the highest expression detected in hemocytes, and the expression level of PmAIF-1 mRNA was significantly up-regulated in hemocytes, gill, digestive gland under bacterial challenge and tissue injury. After challenged by gram-negative bacteria Vibrio alginolyticus and Vibrio parahaemolyticus, gram-positive bacteria Bacillus subtilis, the expression level of this gene in hemocytes were all up-regulated and reached the maximum point at 12h (5.80 folds, P<0.01), 6h (5.02 folds, P<0.01) and 12h (5.49 folds, P<0.01), respectively. Under shell damage and mantle injury, PmAIF-1 mRNA increased gradually in the first 3h and reached a peak of expression at 6h post-injury. These findings suggest that PmAIF-1 is an acute-response protein involved in the innate immune responses of pearl oysters, and provide general information about the mechanisms of innate immune defense against bacterial infection in pearl oysters.

Cloning and characterization of allograft inflammatory factor-1 (AIF-1) from manila clam Venerupis philippinarum

Allograft inflammatory factor-1 (AIF-1) is a 17 kDa interferon-γ-inducible Ca 2+-binding EF-hand protein that plays a significant role not only in different host responses to inflammatory stimuli, but in the whole host immune defense reaction. In the present study, the full-length cDNA of AIF-1 was identified from manila clam Venerupis philippinarum (denoted as VpAIF-1) by cDNA library and RACE approaches. The cDNA of VpAIF-1 consisted of a 5-terminal untranslated region (UTR) of 153 bp, a 3′UTR of 219 bp with a poly (A) tail, and an open reading frame (ORF) of 516 bp encoding a polypeptide of 171 amino acids with the putative molecular mass of 17 kDa. The deduced amino acid of VpAIF-1 shared two EF hand Ca 2+-binding motifs like other AIF-1s. Phylogenetic analysis further indicated that VpAIF-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AIF-1 protein family. Spatial expression analysis indicated that mRNA transcript of VpAIF-1 was found to be most abundantly expressed in the tissues of haemocytes, gills and hepatopancreas, weakly expressed in the tissues of mantle, muscle, and foot. After challenged by Vibrio anguillarum, the mRNA level of VpAIF-1 in overall haemocytes population was recorded by quantitative real-time RT-PCR. VpAIF-1 mRNA was down-regulated in the first 12 h post-infection. Then, the expression level increased to the peak at 72 h and recovered to the 48 h-level at 96 h. All these results indicated that VpAIF-1 was involved in the immune response against microbe infection and might be contributed to the clearance of bacterial pathogens.

Allograft Inflammatory Factor-1 is Up-regulated in Warm and Cold Ischemia-Reperfusion Injury in Rat Liver and May be Inhibited by FK506

Allograft inflammatory factor-1 (AIF-1) plays an important role in immune response and vasculopathy in allografts. The present study investigated activation of AIF-1 in warm and cold ischemia-reperfusion (IR) injury of rat liver, and the potential inhibitory effect of FK506. We used warm IR injury, orthotopic transplantation, and allograft rejection models in this study. We assessed expression of AIF-1 mRNA and protein, as well as its inducers interferon-γ (IFN-γ) and interleukin-1β (IL-1β). The potential inhibitory effect of FK506 on AIF-1 in a rat macrophage cell line and in these three models was also assessed. AIF-1 mRNA and protein, as well as its inducers IFN-γ and IL-1β, were significantly increased in the warm IR injury, orthotopic transplantation, and allograft rejection models. Real-time RT-PCR and Western blotting showed that pretreatment with low-dose FK506 partially inhibited AIF-1 activation as well as its inducers (IFN-γ and IL-1β) in these three models. Western blotting showed that IFN-γ and IL-1β activated AIF-1 in a macrophage cell line, but pretreatment with FK506 did not inhibit AIF-1 activation in vitro. Hematoxylin and eosin staining showed that edema and necrosis in the liver, as well as alanine aminotransferase (ALT) in serum, of these three groups was reduced after FK506 pretreatment. AIF-1 was activated in warm and cold IR injury, and pretreatment with low-dose FK506 partly inhibited AIF-1 activation and reduced warm and cold IR injury.

AIF-1 expression regulates endothelial cell activation, signal transduction, and vasculogenesis

Endothelial cell (EC) activation plays a key role in vascular inflammation, thrombosis, and angiogenesis. Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein that has been implicated in the regulation of inflammation. The expression and function of AIF-1 in EC is uncharacterized, and the purpose of this study was to characterize AIF-1 expression and function in ECs. AIF-1 expression colocalized with CD31-positive ECs in neointima of inflamed human arteries but not normal arteries. AIF-1 is detected at low levels in unstimulated EC, but expression can be increased in response to serum and soluble factors. Stable transfection of AIF-1 small interfering RNA (siRNA) in ECs reduced AIF-1 protein expression by 73% and significantly reduced EC proliferation and migration (P < 0.05 and 0.001). Rescue of AIF-1 expression restored both proliferation and migration of siRNA-expressing ECs, and AIF-1 overexpression enhanced both of these activities, suggesting a strong association between AIF-1 expression and EC activation. Activation of mitogen-activated protein kinase p44/42 and PAK1 was significantly reduced in siRNA ECs challenged with inflammatory stimuli. Reduction of AIF-1 expression did not decrease EC tube-like structure or microvessel formation from aortic rings, but overexpression of AIF-1 did significantly increase the number and complexity of these structures. These data indicate that AIF-1 expression plays an important role in signal transduction and activation of ECs and may also participate in new vessel formation.

Expression of AIF-1 and RANTES in Unexplained Spontaneous Abortion and Possible Association with Alloimmune Abortion

Objective To investigate the effects of allograft inflammatory factor-1 (AIF-1) and (RANTES) in sera and deciduas on unexplained early spontaneous abortion. Methods AIF-1 and RANTES were examined in sera and deciduas/endometria of 43 unexplained early spontaneous abortion women (group A), 40 healthy women with early pregnancy(group B) and 20 healthy women with no pregnancy (group C). Immunohistochemistry and enzyme linked immunosorbent assay (ELISA) were used in this study. Results AIF-1 protein was expressed both in deciduas of group A and in endometria of group C. In group A, H scores in the recurrent abortion deciduas specimens were significantly greater than those in the first abortion;in endometrium, expression of AIF-1 was greater in the secretory than in proliferative phase of group C. In group B, concentrations of RANTES in sera were higher in 7th-8th week of pregnancy than in 6th-7th and >8th week of pregnancy; expression of AIF-1 protein showed a negative correlation with RASNTES concentration; a significant increase of the RANTES levels in sera and tissue was observed in group B. Conclusion These results demonstrate, for the first time, that AIF-1 are expressed in deciduas of unexplained spontaneous abortion suggesting that AIF-1 involve in alloimmune abortion; RANTES might act as a novel blocking antibody;AIF-1 and RANTES might act as reliable markers for diagnosis of early alloimmune abortion.

A Critical Role for Allograft Inflammatory Factor-1 in the Pathogenesis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is characterized by massive synovial proliferation, angiogenesis, subintimal infiltration of inflammatory cells and the production of cytokines such as TNF-alpha and IL-6. Allograft inflammatory factor-1 (AIF-1) has been identified in chronic rejection of rat cardiac allografts as well as tissue inflammation in various autoimmune diseases. AIF-1 is thought to play an important role in chronic immune inflammatory processes, especially those involving macrophages. In the current work, we examined the expression of AIF-1 in synovial tissues and measured AIF-1 in synovial fluid (SF) derived from patients with either RA or osteoarthritis (OA). We also examined the proliferation of synovial cells and induction of IL-6 following AIF-1 stimulation. Immunohistochemical staining showed that AIF-1 was strongly expressed in infiltrating mononuclear cells and synovial fibroblasts in RA compared with OA. Western blot analysis and semiquantitative RT-PCR analysis demonstrated that synovial expression of AIF-1 in RA was significantly greater than the expression in OA. AIF-1 induced the proliferation of cultured synovial cells in a dose-dependent manner and increased the IL-6 production of synovial fibroblasts and PBMC. The levels of AIF-1 protein were higher in synovial fluid from patients with RA compared with patients with OA (p < 0.05). Furthermore, the concentration of AIF-1 significantly correlated with the IL-6 concentration (r = 0.618, p < 0.01). These findings suggest that AIF-1 is closely associated with the pathogenesis of RA and is a novel member of the cytokine network involved in the immunological processes underlying RA.

Expression of allograft inflammatory factor-1 in kidneys: A novel molecular component of podocyte

Our comprehensive gene expression profiles of the kidneys in an anti-glomerular basement membrane (GBM) nephritis model using DNA arrays revealed that allograft inflammatory factor-1 (AIF-1) was one of the highly expressed genes. Here, we explored the pathological significance of AIF-1 expression in the kidneys. The expression pattern of AIF-1 mRNA and protein in the kidneys of normal and diseased rats, such as anti-GBM nephritis and puromycin aminonucleoside nephrosis, was investigated by in situ hybridization, immunohistochemistry, and immunoelectron microscopy. Furthermore, the expression of AIF-1 in human kidneys and urinary sediments was examined. AIF-1 was expressed at both mRNA and protein levels in podocytes of normal and diseased rats, and in infiltrating cells in anti-GBM nephritis kidneys. The expression of AIF-1 in podocytes was constitutive; positive in podocytes of both normal and diseased rats. In humans, AIF-1 was expressed in podocytes and infiltrating inflammatory cells, similarly. Moreover, it was detected in urinary podocytes from patients with immunoglobulin A nephropathy. These data document for the first time that AIF-1, a constitutively expressed protein in rat and human podocytes, is a novel molecular component of podocytes, and that the upregulation of AIF-1 in an anti-GBM nephritis model may mainly be a consequence of its expression in infiltrating cells.

Expression of allograft inflammatory factor 1 in tissues from patients with systemic sclerosis and in vitro differential expression of its isoforms in response to transforming growth factor β

Allograft inflammatory factor 1 (AIF-1), a protein initially identified in chronically rejected rat cardiac allografts, is involved in the immune response and proliferative vasculopathy that occurs during allograft rejection. Three well-characterized isoforms of AIF-1 result from alternative messenger RNA (mRNA) splicing. We previously identified a strong association of systemic sclerosis (SSc) with a polymorphism in AIF-1 isoform 2. The purpose of this study was to investigate AIF-1 expression in affected tissues from patients with SSc and to examine the regulation of its isoforms by transforming growth factor beta (TGFbeta). AIF-1 in the skin and lung tissues of patients with SSc was analyzed by immunochemistry. AIF-1 isoform expression in response to TGFbeta and interferon-gamma stimulation was examined by quantitative polymerase chain reaction (PCR). AIF-1 protein was present in affected vessels of the lung and skin lesions of patients with SSc. Quantitative PCR showed an average of 14-fold higher mRNA levels in affected SSc skin than in normal skin. Double-label immunofluorescence staining demonstrated that T cells, macrophages, and endothelial cells in affected tissues expressed AIF-1. Stimulation of peripheral blood mononuclear cells with TGFbeta caused a specific and significant increase in the expression of AIF-1 isoform 2 transcripts (P < 0.005), which was due to stabilization of AIF-1 isoform 2 mRNA. These data suggest that AIF-1 plays an important role in the pathogenesis of SSc owing to its increased expression in affected tissues and to the specific stimulation of AIF-1 isoform 2 by TGFbeta.

Inhibition of AIF-1 expression by constitutive siRNA expression reduces macrophage migration, proliferation, and signal transduction initiated by atherogenic stimuli

Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein. Several studies have reported increased AIF-1 expression in activated macrophages and have implicated AIF-1 as a marker of activated macrophages. However, the function of AIF-1 in macrophages and the mechanism whereby it participates in macrophage activation are unknown at this time. Immunohistochemical analysis colocalized AIF-1 expression with CD68-positive macrophages in atherosclerotic human coronary arteries. Subsequent experiments were designed to determine a role for AIF-1 in macrophage activation in response to atherogenic stimuli. Stimulation of human and murine macrophages with oxidized LDL significantly increased AIF-1 expression above basal levels. Stable transfection of AIF-1 small interfering RNA (siRNA) in macrophages reduced AIF-1 protein expression by 79% and reduced macrophage proliferation by 52% (P < 0.01). Inhibition of proliferation was not due to induction of apoptosis. Sequences that did not knock down AIF-1 expression had no effect on proliferation. AIF-1 siRNA expression reduced macrophage migration by 60% (P < 0.01). Both proliferation and migration of siRNA-expressing macrophages could be restored by adenoviral expression of AIF-1 (P < 0.001 and 0.005, respectively), suggesting a tight association between AIF-1 expression and macrophage activation. Phosphorylation of Akt, p44/42 MAPK, and p38 kinase were significantly reduced in siRNA macrophages challenged with oxidized LDL (P < 0.05). Phosphorylation of p38 kinase was significantly inhibited in siRNA macrophages stimulated with T lymphocyte conditioned medium (P < 0.05). These data indicate that AIF-1 mediates atherogenesis-initiated signaling and activation of macrophages.

Expression of Allograft Inflammatory Factor-1 in Human Eutopic Endometrium and Endometriosis: Possible Association with Progression of Endometriosis

Allograft inflammatory factor-1 (AIF-1) is a cytokine originally identified in rat cardiac allografts with chronic rejection. AIF-1 is expressed in various human immune-related tissues and is thought to play a role in inflammatory responses and the immune activation and function of macrophages. Expression has also been shown in human placentas and bovine embryos, suggesting that AIF-1 may be involved in reproductive function. Immune factors are thought to be involved in the pathogenesis of endometriosis. High concentrations of activated macrophages and various cytokines have been found in the peritoneal fluid of patients with endometriosis. In the current work we examined the expression of AIF-1 in human eutopic endometrium and endometriosis, and measured AIF-1 in peritoneal fluid samples from women with and without endometriosis. RT-PCR, Western blot analysis, and immunohistochemistry showed that AIF-1 mRNA and protein were expressed both in eutopic endometrium and in endometriotic tissue. In eutopic endometrium, expression was greater in the late secretory and menstrual phases than in other phases of the menstrual cycle (P < 0.01). AIF-1 protein was present in greater amounts in peritoneal fluid from patients with endometriosis than in women without it (P < 0.01), and its concentration correlated with the Revised American Society for Reproductive Medicine score (rs = 0.693; P < 0.0001). Peritoneal macrophages from endometriosis patients secreted more AIF-1 than those from unaffected women (P < 0.05). AIF-1 release from macrophages was stimulated by IL-1beta (P < 0.01) and interferon-gamma (P < 0.05). These results demonstrate for the first time that AIF-1 is expressed in eutopic endometrium and endometriotic tissue, suggesting that AIF-1 is one cytokine in the local network involved in the onset of menstruation. AIF-1 derived from peritoneal macrophages may also possibly play a significant role in the pathophysiology and progression of endometriosis.

Expression of allograft inflammatory factor-1 is a marker of activated human vascular smooth muscle cells and arterial injury.

The cytokine-induced activation and proliferation of medial vascular smooth muscle cells (VSMCs) leading to intimal hyperplasia is one of the most critical cellular events in the formation of transplant arteriopathy and balloon angioplasty-induced restenosis. Allograft inflammatory factor-1 (AIF-1) is a calcium-binding protein that we have previously shown to be expressed in balloon angioplasty-injured rat carotid arteries. We hypothesized that AIF-1 expression may be associated with the VSMC response to injury. In this study, we examined AIF-1 expression in immunologic and mechanical models of arterial injury. Reverse transcription-polymerase chain reaction and Western analysis demonstrated that AIF-1 is acutely and transiently expressed in aortic medial smooth muscle cells of rat cardiac allografts, with mRNA and protein peaking at 3 to 7 days after transplant and declining by 10 days after transplant. Immunohistochemical analysis identified abundant AIF-1 in the medial VSMCs of these vessels. Immunohistochemical analysis of balloon angioplasty-injured swine coronary arteries also demonstrates an acute AIF-1 expression detectable by 24 hours and continuing up to 14 days after the procedure. AIF-1 in these vessels also localizes to the medial VSMCs and cells of the developing neointima. AIF-1 protein is not expressed in quiescent cultured human VSMCs but is induced in cells challenged with various inflammatory cytokines, primarily by interferon-gamma, interleukin-1beta, and T-cell-conditioned media. Transfection and overexpression of AIF-1 in human VSMCs result in enhanced growth of these cells. Taken together, these data indicate that AIF-1 expression is associated with vascular trauma and suggest that this protein may play a role in VSMC activation subsequent to arterial injury.

Isolation of two novel alternative splicing variants of allograft inflammatory factor-1.

Allograft inflammatory factor-1 (AIF-1) was initially cloned from rat cardiac allografts undergoing chronic rejection. AIF-1, possessing an EF-hand-like motif, is thought to be a Ca2+ binding protein. In this study, we identified two novel alternatively spliced variants of AIF-1 by reverse-transcription polymerase chain reaction. One variant encodes an AIF-1 protein that lacks 14 amino acids corresponding to one exon. The other variant encodes a truncated AIF-1 protein due to a frameshift introduced by an 85-bp insertion, and its C-terminal region differs from that of AIF-1. Interestingly, these variants have incomplete and no EF-handlike motifs, respectively. The expression level of the latter variant is high in peripheral blood leukocytes, and it is selectively expressed in macrophage-like cell lines.

Cloning and characterization of allograft inflammatory factor-1: a novel macrophage factor identified in rat cardiac allografts with chronic rejection.

The development of arteriosclerotic lesions in the Lewis to F344 rat model of chronic cardiac rejection is characterized by macrophage adhesion to the vessel lumen and macrophage infiltration in the neointima prior to smooth muscle cell accumulation. We report the cloning and characterization of allograft inflammatory factor-1 (AIF-1), a novel cDNA that is expressed early and persistently in chronically rejecting cardiac allografts but is absent in cardiac syngrafts and host hearts. The full-length cDNA codes for a hydrophilic polypeptide of 17 kD that contains a 12-amino acid region similar to an EF-hand (calcium-binding) domain. In cardiac allografts AIF-1 transcripts and protein localized to infiltrating mononuclear cells. Analysis of isolated cell populations confirmed that AIF-1 was selectively expressed in macrophages and neutrophils and demonstrated that AIF-1 transcripts could be upregulated by sixfold after stimulation with the T cell-derived cytokine IFN-gamma. Treatment with a diet deficient in essential fatty acids (which attenuates arteriosclerosis) or CTLA-4 Ig (which blocks lymphocyte activation) significantly decreased AIF-1 transcript levels. Upregulation of AIF-1 in the setting of T cell activation suggests that it may play a role in macrophage activation and function.