Guanylate Binding Protein-1 Research Articles

Abstract A4: Guanylate binding protein-1 promotes glioma cell invasion through induction of MMP1 expression

Abstract Glioma is the most common and deadly human brain tumor. Epidermal growth factor receptor (EGFR) amplification or constitutively active mutation (EGFRVIII) is one of the most frequent genetic lesions in gliomas. To identify target genes regulated by EGFR signaling, we performed expression array analysis and found that human guanylate binding protein-1 (GBP-1), a large GTPase that is induced by inflammatory cytokines, is one of the predominant genes induced by EGFR/EGF or EGFR VIII kinase activity. Our results demonstrate that EGF/EGFR signaling could dramatically induce Gbp-1 expression at both the mRNA and protein level through the p38 kinase signaling cascade that is distinct from Stat1 signaling used by interferon gamma. Interestingly, we show that Gbp-1 is required for EGFR-mediated matrix metallopeptidase-1 (MMP1) expression, and plays a role in glioma cell invasion in vitro. Overexpression of Gbp-1 induced MMP1 expression in glioma cells, which was independent of Gbp-1 GTPase activity. In contrast, siRNA-mediated knockdown of Gbp-1 inhibited MMP1 expression and in turn blocked cell invasion as well. Consistently, we found that GBP-1 is overexpressed and positively correlated with EGFR/EGFRVIII and MMP1 expression in human glioma clinical samples and glioma cell lines. Therefore, this study suggests that Gbp-1 may be a novel therapeutic target in glioma. Citation Information: Cancer Res 2009;69(23 Suppl):A4.

Antiviral effects of the interferon-induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein

Interferons (IFNs) and the interferon-stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We have reported previously that ISGs, including guanylate binding protein 1 (GBP-1), interferon alpha inducible protein (IFI)-6-16, and IFI-27, inhibit HCV subgenomic replication. In this study we investigated the effects of these ISGs against HCV in cell culture and their direct molecular interaction with viral proteins. HCV replication and virus production were suppressed significantly by overexpression of GBP-1, IFI-6-16, or IFI-27. Knockdown of the individual ISGs enhanced HCV RNA replication markedly. A two-hybrid panel of molecular interaction of the ISGs with HCV proteins showed that GBP-1 bound HCV-NS5B directly. A protein truncation assay showed that the guanine binding domain of GBP-1 and the finger domain of NS5B were involved in the interaction. Binding of NS5B with GBP-1 inhibited its guanosine triphosphatase GTPase activity, which is essential for its antiviral effect. Taken together, interferon-induced GBP-1 showed antiviral activity against HCV replication. Binding of the HCV-NS5B protein to GBP-1 countered the antiviral effect by inhibition of its GTPase activity. These mechanisms may contribute to resistance to innate, IFN-mediated antiviral defense and to the clinical persistence of HCV infection.

Interferon-α counteracts the angiogenic switch and reduces tumor cell proliferation in a spontaneous model of prostatic cancer

Interferon (IFN)-alpha is a cytokine with marked therapeutic activity in transplantable tumor models, that is in part due to angiogenesis inhibition. Aim of this study was to investigate the effects of IFN-alpha during the early phases of tumor development in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. To provide sustained IFN-alpha production, TRAMP mice were injected intraperitoneally with lentiviral vectors. IFN-alpha administration resulted in rapid and protracted upregulation of IFN-alpha-regulated genes associated with antiangiogenic and antiproliferative functions in the prostate of TRAMP mice, including guanylate-binding protein 1 (GBP-1), IFI204 and CXCL10-11. These transcriptional changes were accompanied by effects on the tumor vasculature, including significant reduction of intraductal microvessel density and increased pericyte coverage, and marked reduction of tumor cell proliferation, without induction of tumor necrosis. Intriguingly, GBP-1 and myxovirus resistance A, two IFN-regulated proteins, were found expressed in approximately 40% of human prostate cancer samples analyzed, suggesting expression of endogenous IFN-alpha. Overall, these findings demonstrate that IFN-alpha is able to counteract the angiogenic switch and impairs tumor cell proliferation in preinvasive lesions. Since the angiogenic switch also marks progression of human prostatic cancer, these results highlight the potential of angiogenesis inhibitors for the development of chemoprevention strategies in high-risk individuals.

Guanylate Binding Protein-1 GTPase (Gbp1) Inhibits Ragweed Extract (RWE) Induced Th 2 Cytokine Production from CD4T-Cells Cultured with BMDC

Guanylate Binding Protein-1 GTPase (Gbp1) Inhibits Ragweed Extract (RWE) Induced Th 2 Cytokine Production from CD4T-Cells Cultured with BMDC

Role of Individual Domains and Identification of Internal Gap in Human Guanylate Binding Protein-1

Unlike other GTPases, interferon-gamma-induced human guanylate binding protein-1 has the ability to hydrolyze GTP to both GDP and GMP, with GMP being the major product of the reaction. This protein has two domains, an N-terminal globular domain and a C-terminal helical domain. These two domains are connected by a short intermediate region consisting of a two-stranded β-sheet and a helix. As human guanylate binding protein-1 has been shown to undergo stimulated GTPase activity without external GTPase-activating protein, we sought to understand the roles of each of the two individual domains, the intermediate region, a conserved motif ( 103DXEKGD 108), and the mechanism of the stimulation of GTPase activity. The steady-state assays using radiolabeled [α- 32P]GTP on the wild-type protein suggest that the stimulation of activity primarily occurs during the cleavage of the second phosphate of GTP rather than the first, through allosteric interaction. Using several truncated and mutant proteins, we demonstrate for the first time that both the α-helix of the intermediate region and the 103DXEKGD 108 motif play critical roles for the hydrolysis to GMP, but they appear to act in different ways: α-helix acts through structural stabilization by allosteric interaction and, thus, acts as an internal GTPase-activating protein, whereas the motif might act by providing necessary catalytic residues. Our data also show that the N-terminal globular domain is able to perform only the first catalysis (GTP to GDP, an activity associated with basal level), but the helical domain in the full-length protein stimulates the hydrolysis of GTP to GMP with higher GMP formation by preventing the dissociation of GDP-bound enzyme dimer.

Guanylate-binding protein-1 is expressed at tight junctions of intestinal epithelial cells in response to interferon-γ and regulates barrier function through effects on apoptosis

Guanylate-binding protein-1 (GBP-1) is an interferon inducible large GTPase involved in endothelial cell proliferation and invasion. In this report, expression and function of GBP-1 were investigated in vitro in intestinal epithelia after exposure to interferon-γ and in human colonic mucosa from individuals with inflammatory bowel disease (IBD). Interestingly, in contrast to other epithelia, GBP-1 distributed to the plasma membrane in intestinal epithelial cells where it colocalized with the tight junction protein coxsackie- and adenovirus receptor. In addition, expression of GBP-1 was upregulated in colonic epithelia of individuals with IBD. Downregulation of GBP-1 by siRNA resulted in enhanced permeability that correlated with increased apoptosis. Indeed, inhibition of caspase activity prevented the inhibition of barrier formation induced by the loss of GBP-1. These data suggest that GBP-1 is a novel marker of intestinal mucosal inflammation that may protect against epithelial apoptosis induced by inflammatory cytokines and subsequent loss of barrier function.

Angiostatic immune reaction in colorectal carcinoma: Impact on survival and perspectives for antiangiogenic therapy

Angiogenesis and inflammation are the 2 major stroma reactions in colorectal carcinoma (CRC). Guanylate binding protein-1 (GBP-1) is a key mediator of angiostatic effects of inflammation. Therefore, we hypothesized that GBP-1 may be a biomarker of intrinsic angiostasis associated with an improved outcome in CRC patients. GBP-1 was strongly expressed in endothelial cells and immune cells in the desmoplastic stroma of 32% of CRC as determined by immunohistochemical investigation of 388 sporadic CRC. Cancer-related 5-year survival was highly significant (p < 0.001) increased (16.2%) in patients with GBP-1-positive CRC. Multivariate analysis showed that GBP-1 is an independent prognostic factor indicating a reduction of the relative risk of cancer-related death by the half (p = 0.032). A comparative transcriptome analysis (22,215 probe sets) of GBP-1-positive (n = 12) and -negative (n = 12) tumors showed that particularly IFN-gamma-induced genes including the major antiangiogenic chemokines CXCL9, CXCL10 and CXCL11 were coexpressed with GBP-1. Altogether our findings indicated that GBP-1 may be a novel biomarker and an active component of a Th-1-like angiostatic immune reaction in CRC. This reaction may affect patient's response to antiangiogenic therapy and the identification of such tumors may provide a novel criterion for patient selection. Moreover, the induction of a Th-1-like angiostatic immune reaction may be a promising approach for the clinical treatment of CRC.

Guanylate binding protein‐1 inhibits spreading and migration of endothelial cells through induction of integrin α4expression

Human guanylate binding protein-1 (GBP-1) is a large GTPase that is induced by inflammatory cytokines and acts antiangiogenically through the inhibition of endothelial cell proliferation and migration. In this study, we detected that GBP-1-expressing cells show a significantly reduced spreading and migration on fibronectin matrices. Investigating possible mechanisms of these effects, we found that integrin alpha(4) (ITGA4) was consistently up-regulated at both the RNA and protein level in GBP-1-expressing cell cultures. Inhibition of cell spreading and migration by GBP-1 was dependent on the binding of ITGA4 to fibronectin. The inflammatory cytokines IL-1beta and TNF-alpha induced ITGA4 expression in HUVECs and inhibited spreading and migration. Knockdown of GBP-1 by shRNA abrogated inflammatory cytokine induced ITGA4 expression and restored spreading and migration capabilities of the cells. These results show that inhibition of endothelial cell spreading and migration by inflammatory cytokines is mediated by GBP-1 through induction of ITGA4 expression. Endothelial cell migration is a key process during angiogenesis. Therefore, ITGA4 may be a novel molecular target to modulate angiogenesis in human disease.

Guanylate binding protein‐1 (GBP‐1) is upregulated in intestinal epithelia under inflammatory conditions, localizes to tight junctions and regulates epithelial barrier function

GBP‐1 belongs to the family of large GTPases, is strongly induced in leukocytes and endothelial cells after IFN‐γ treatment and regulates endothelial cell proliferation under inflammatory conditions. Given the dependence of GBP‐1 expression on IFN‐γ, and the elevated level of IFN‐γ within intestinal mucosa of individuals with inflammatory bowel disease (IBD), we investigated whether GBP‐1 is expressed in colonic epithelial cells. Analyses of different epithelial cell lines revealed that GBP‐1 expression was low to absent in all cells tested under resting conditions, but was strongly induced in all tested epithelial cell lines after IFN‐γ treatment. Localization studies revealed that GBP‐1 is expressed at the plasma membrane and colocalized with the tight junction protein coxsackie‐ and adenovirus receptor (CAR) only in colonic epithelial cells. Immunofluorescence labeling of human colonic mucosa revealed upregulation of GBP‐1 expression in the epithelium of individuals with active IBD. Intriguingly, colocalization of GBP‐1 with CAR was also observed in IBD patients. Functional studies employing siRNA‐mediated knock down of GBP‐1 in colonic epithelia demonstrated delayed epithelial barrier formation and increased susceptibility to apoptosis. These data suggest that GBP‐1 expression in colonic epithelia may play a role in regulation of the epithelial barrier under inflammatory conditions.

Antiviral effect of interferon-induced guanylate binding protein-1 against Coxsackie virus and Hepatitis B virus B3 in Vitro

Guanylate binding protein-1(GBP-1) is an interferon-induced protein. To observe its antiviral effect against Hepatitis B virus (HBV) and Coxsackie virus B3 (CVB3), we constructed an eukaryotic expression vector of human GBP-1(hGBP-1). Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector, then subcloned into a pCDNA3.1(-) vector. Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells, and inhibition effect of hGBP-1 against HBV replication was observed. Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3, and viral yield in cultures were detected. The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells. hGBP-1 inhibit CVB3 but not HBV replication in vitro . These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.

Interferon-α prevents apoptosis of endothelial cells after short-term exposure but induces replicative senescence after continuous stimulation

Although the antiangiogenic activity of type I interferons (IFN) is well known, the mechanism by which it occurs is unclear. In the present study, we have investigated effects of short-term and long-term IFN-α exposure on different types of endothelial cells (EC). Short-term IFN-α treatment resulted in a distinct reduction of apoptosis of serum and growth factor starved HUVEC and HDMEC. This was accompanied by a strong upregulation of the IFN inducible guanylate binding protein-1 (GBP-1) whereas no consistent regulation of several known antiapoptotic proteins was evident. Stable transfection of HUVEC with an expression vector for GBP-1 mimicked the protective effect of IFN-α, suggesting that GBP-1 may contribute to the inhibition of apoptosis. When IFN-α, together with serum and EC growth factors, was present continuously a decrease of population doublings by more than 40% was observed in both HDMEC and HCAEC. In addition, the cells displayed a senescent phenotype significantly earlier than control cells and showed an increased adherence for monocytes. Our findings suggest that the antiangiogenic effect of IFN-α is mediated by inducing EC senescence rather than EC apoptosis. Furthermore IFN-α released in chronic inflammatory conditions might contribute via its prosenescent activity to the pathogenesis of atherosclerosis.

Human Guanylate Binding Protein-1 Is a Secreted GTPase Present in Increased Concentrations in the Cerebrospinal Fluid of Patients with Bacterial Meningitis

Interferon-gamma-induced GTPases are key to the protective immunity against microbial and viral pathogens. As yet, the cell interior has been regarded as the exclusive residence of these proteins. Here we show that a member of this group, human guanylate binding protein-1 (hGBP-1), is secreted from cells. Secretion occurred in the absence of a leader peptide via a nonclassical, likely ABC transporter-dependent, pathway, was independent of hGBP-1 GTPase activity and isoprenylation, and did not require additional interferon-gamma-induced factors. Interestingly, hGBP-1 was only secreted from endothelial cells but not from any of the nine different cell types tested. Clinically most important was the detection of significantly (P<0.001, Mann-Whitney U-test) increased hGBP-1 concentrations in the cerebrospinal fluid of patients with bacterial meningitis (n=32) as compared to control patients (n=74). In this first report of a secreted GTPase, we demonstrate that secreted hGBP-1 may be a useful surrogate marker for diagnosis of bacterial meningitis.

Mycobacterium tuberculosisandMycobacterium avium InhibitIFN-γ-Induced Gene Expression by TLR2-Dependent and Independent Pathways

Mycobacteria-infected macrophages are poor responders to interferon-gamma (IFN-gamma), resulting in decreased expression of IFN-gamma-induced genes. In the present study, we examined the inhibition of IFN-gamma-induced gene expression by Mycobacterium tuberculosis and four different Mycobacterium avium strains in mouse RAW264.7 macrophages. Gamma-irradiated M. tuberculosis inhibited mRNA expression of a panel of six different IFN- gamma-induced genes. All four of the M. avium strains completely inhibited IFN-gamma-induced expression of MHC class II Aalpha and Ebeta mRNA. However, the Mac101 strain, which is serovar 1, inhibited IFN-gamma induction of IFN regulatory factor-1 (IRF-1) and guanylate-binding protein-1 (GBP-1) mRNA to a greater extent than the other M. avium strains, which are serovar 2. In this study, we also show that mycobacteria inhibit gene expression by both toll-like receptor 2 (TLR2)-dependent and independent pathways. The inhibition of IFN-gamma-induced gene expression by M. avium was reduced but not completely blocked in macrophages from TLR2(/) mice. IFN-gamma-induced gene expression was also inhibited by mycobacteria in RAW264.7 cells expressing dominantnegative TLR2 or myeloid differentiation factor 88 (MyD88), further indicating the existence of a pathway independent of TLR2 and MyD88. These data suggest that mycobacteria inhibit IFN-gamma-induced gene expression by multiple pathways involving both TLR2 and non-TLR receptors.

GBP1 overexpression is associated with a paclitaxel resistance phenotype

In the search for novel genes involved in the paclitaxel resistance phenotype, prior studies of gene expression in paclitaxel-resistant cell lines and their paired drug-sensitive parental lines using high-density Affymetrix GeneChip arrays identified guanylate-binding protein 1 (GBP1) gene as an overexpressed transcript. The GBP1 gene encodes a large GTPase that is induced by interferon gamma (IFN-gamma) in a variety of eukaryotic cells. In this report we characterize GBP1 and demonstrate that GBP1 expression is consistently upregulated in 7 of 8 paclitaxel or doxorubicin-resistant human cancer cell lines as compared to its expression in the relevant drug-sensitive parental lines. Analysis of GBP1 expression using the Cancer Profiling Array showed that GBP1 is ubiquitously expressed with no significant difference in expression levels between normal and tumor tissue. Parallel analysis of the Cancer Cell Line Profiling Array determined that GBP1 expression in a majority of cell lines derived from human tumors of different tissue origin was induced to variable levels following exposure to multiple stress agents including paclitaxel and doxorubicin. Importantly, stable expression of a GBP1 transgene in the paclitaxel-sensitive ovarian cancer cell line OVCAR8 was sufficient to confer moderate paclitaxel resistance. Our data suggest that increased expression of the GBP1 gene may play an important role in the development of multi-drug resistance (MDR).

SUMO-1 conjugation selectively modulates STAT1-mediated gene responses

Signal transducers and activators of transcription 1 (STAT1) is a critical mediator of interferon (IFN)-induced gene responses. Recently, STAT1 was found to become modified by small ubiquitin-like modifier 1 (SUMO-1) conjugation at Lys703 through the SUMO E3 ligase function of protein inhibitors of activated STAT (PIAS) proteins. However, the physiologic function of sumoylation in STAT1 is still unclear. Here, we show that mutations in the SUMO attachment site in STAT1 result in increased transcriptional activity in a fashion that is selective among IFN-gamma target genes. The sumoylation-defective STAT1 mutant displayed increased induction of guanylate-binding protein 1 (GBP1) and transporters associated with antigen presentation 1 (TAP1) transcription but not interferon regulatory factor 1 (IRF1) transcription. Moreover, the sumoylation-defective mutant STAT1-KR showed a prolonged DNA-binding activity and nuclear localization in response to IFN-gamma stimulation. These results suggest that sumoylation has a defined negative regulatory effect on selective STAT1-mediated transcription responses.

Human guanylate binding protein-1 (hGBP-1) characterizes and establishes a non-angiogenic endothelial cell activation phenotype in inflammatory diseases

Blood vessel activation in inflammatory diseases is triggered by a myriad of different factors that partially reveal opposite activities on endothelial cells (EC). For example, inflammatory cytokines (IC) inhibit EC proliferation and induce cell adhesiveness for leukocytes. In contrast, angiogenic growth factors (AGF) activate EC proliferation and inhibit cell adhesiveness for leukocytes. In consequence, IC and AGF may induce two different activation phenotypes in EC that appear in a temporally and/or spatially coordinated manner in inflammatory tissues. Human guanylate binding protein-1 (hGBP-1) is a member of the large GTPase protein family. New results demonstrate that hGBP-1 is a specific marker of IC-activated EC that allows to differentiate the IC- and AGF-activated phenotype of EC at the single cell level, both in vitro and in vivo. In addition, hGBP-1 is the key mediator of the inhibitory effects of IC on EC proliferation and invasiveness. Both the expression pattern of hGBP-1 and its activity in EC supported the hypothesis that IC- and AGF-activation induce distinct adversely related phenotypes in EC. In future, hGBP-1 may be used as a marker to monitor the IC-induced phenotype of EC in inflammation and may also be exploited as a target to modulate EC activity in inflammatory diseases and tumor angiogenesis.

Nuclear factor-kappaB motif and interferon-alpha-stimulated response element co-operate in the activation of guanylate-binding protein-1 expression by inflammatory cytokines in endothelial cells.

The large GTPase GBP-1 (guanylate-binding protein-1) is a major IFN-gamma (interferon-gamma)-induced protein with potent anti-angiogenic activity in endothelial cells. An ISRE (IFN-alpha-stimulated response element) is necessary and sufficient for the induction of GBP-1 expression by IFN-gamma. Recently, we have shown that in vivo GBP-1 expression is strongly endothelial-cell-associated and is, in addition to IFN-gamma, also activated by interleukin-1beta and tumour necrosis factor-alpha, both in vitro and in vivo [Lubeseder-Martellato, Guenzi, Jörg, Töpolt, Naschberger, Kremmer, Zietz, Tschachler, Hutzler, Schwemmle et al. (2002) Am. J. Pathol. 161, 1749-1759; Guenzi, Töpolt, Cornali, Lubeseder-Martellato, Jörg, Matzen, Zietz, Kremmer, Nappi, Schwemmle et al. (2001) EMBO J. 20, 5568-5577]. In the present study, we identified a NF-kappaB (nuclear factor kappaB)-binding motif that, together with ISRE, is required for the induction of GBP-1 expression by interleukin-1beta and tumour necrosis factor-alpha. Deactivation of the NF-kappaB motif reduced the additive effects of combinations of these cytokines with IFN-gamma by more than 50%. Importantly, NF-kappaB p50 rather than p65 activated the GBP-1 promoter. The NF-kappaB motif and ISRE were detected in an almost identical spatial organization, as in the GBP-1 promoter, in the promoter regions of various inflammation-associated genes. Therefore both motifs may constitute a cooperative inflammatory cytokine response module that regulates GBP-1 expression. Our findings may open new perspectives for the use of NF-kappaB inhibitors to support angiogenesis in inflammatory diseases including ischaemia.

Molecular engineering of a backwards-moving myosin motor.

All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin- and nucleotide-binding sites and produces force and movement. The light-chain-binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm. Myosins move on polarized actin filaments either forwards to the barbed (+) or backwards to the pointed (-) end. Here, we describe the engineering of an artificial backwards-moving myosin from three pre-existing molecular building blocks. These blocks are: a forward-moving class I myosin motor domain, a directional inverter formed by a four-helix bundle segment of human guanylate-binding protein-1 and an artificial lever arm formed by two alpha-actinin repeats. Our results prove that reverse-direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180 degrees.

The guanylate binding protein-1 GTPase controls the invasive and angiogenic capability of endothelial cells through inhibition of MMP-1 expression.

Expression of the large GTPase guanylate binding protein-1 (GBP-1) is induced by inflammatory cytokines (ICs) in endothelial cells (ECs), and the helical domain of the molecule mediates the repression of EC proliferation by ICs. Here we show that the expression of GBP-1 and of the matrix metalloproteinase-1 (MMP-1) are inversely related in vitro and in vivo, and that GBP-1 selectively inhibits the expression of MMP-1 in ECs, but not the expression of other proteases. The GTPase activity of GBP-1 was necessary for this effect, which inhibited invasiveness and tube-forming capability of ECs in three-dimensional collagen-I matrices. A GTPase-deficient mutant (D184N-GBP-1) operated as a transdominant inhibitor of wild-type GBP-1 and rescued MMP-1 expression in the presence of ICs. Expression of D184N-GBP-1, as well as paracrine supplementation of MMP-1, restored the tube-forming capability of ECs in the presence of wild-type GBP-1. The latter finding indicated that the inhibition of capillary formation is specifically due to the repression of MMP-1 expression by GBP-1, and is not affected by the anti-proliferative activity of the helical domain of GBP-1. These findings substantiate the role of GBP-1 as a major regulator of the anti-angiogenic response of ECs to ICs.

Guanylate-Binding Protein-1 Expression Is Selectively Induced by Inflammatory Cytokines and Is an Activation Marker of Endothelial Cells during Inflammatory Diseases

During angiogenesis and inflammatory processes, endothelial cells acquire different activation phenotypes, whose identification may help in understanding the complex network of angiogenic and inflammatory interactions in vivo. To this goal we investigated the expression of the human guanylate-binding protein (GBP)-1 that is highly induced by inflammatory cytokines (ICs) and, therefore, may characterize IC-activated cells. Using a new rat monoclonal antibody raised against GBP-1, we show that GBP-1 is a cytoplasmic protein and that its expression in endothelial cells is selectively induced by interferon-gamma, interleukin-1alpha, interleukin-1beta, or tumor necrosis factor-alpha, but not by other cytokines, chemokines, or growth factors. Moreover, we found that GBP-1 expression is highly associated with vascular endothelial cells as confirmed by the simultaneous detection of GBP-1 and the endothelial cell-associated marker CD31 in a broad range of human tissues. Notably, GBP-1 expression was undetectable in the skin, but it was highly induced in vessels of skin diseases with a high-inflammatory component including psoriasis, adverse drug reactions, and Kaposi's sarcoma. These results indicate that GBP-1 is a novel cellular activation marker that characterizes the IC-activated phenotype of endothelial cells.