The neurosensory tachykinins substance P and neurokinin A directly induce keratinocyte nerve growth factor.

Nerve growth factor is an essential neurotrophic factor required for the growth and maintenance of cutaneous sensory nerves. In the skin, keratinocytes are a significant source of nerve growth factor; however, the regulation of cutaneous nerve growth factor production still remains to be fully understood. In this study we tested the hypothesis that neuropeptides released by cutaneous sensory nerves have the capacity to modulate directly the expression of keratinocyte nerve growth factor, which would have important implications for the maintenance and repair of nerves in the skin. In order to address this question experimentally we examined the effect of the neuropeptides, substance P and neurokinin A, on nerve growth factor expression in human keratinocytes and the murine keratinocyte PAM 212 cell line by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and the PC-12 nerve growth factor bioassay. The results of these studies indicated that substance P and neurokinin A can directly induce nerve growth factor mRNA expression and the secretion of bioactive nerve growth factor protein in both human and murine keratinocytes. The specificity of these responses was demonstrated using neuropeptide receptor antagonists and nerve growth factor blocking antibodies. Additional studies also demonstrated a significant in vivo upregulation of keratinocyte nerve growth factor expression in murine epidermis after the topical application of the neuropeptide releasing agent capsaicin. This is the first report demonstrating the induction of cutaneous nerve growth factor by sensory nerve-derived neuropeptides such as substance P and neurokinin A. This direct effect of the neurosensory system on keratinocyte nerve growth factor production may have important consequences for the maintenance and regeneration of cutaneous nerves in normal skin and during inflammation and wound healing.

Interleukin-18 expression and modulation in human corneal epithelial cells.

PURPOSE: The interferon-gamma-inducing factor Interleukin-18 (IL-18) is a recently described cytokine that appears to have multiple important pro-inflammatory effects including the induction of interferon-gamma (IFN-gamma) by activated T-cells. The expression of IL-18 by human cornea has not been previously reported. In the present study, we examine the possibility that human corneal epithelial cells are capable of producing this leukocyte-activating factor which may play an important role in IFN-gamma-dependent inflammation responses in the cornea. METHODS: Northern blot analysis and ELISA were used to investigate the in vitro expression of IL-18 mRNA and protein respectively in primary (HCEC) and transformed human corneal epithelial cells (HCET). To determine if IL-18 expression was modulated by pro-inflammatory mediators, cells were treated with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA) or synthetic double stranded RNA (poly dI : dC). IL-18 bioactivity was determined in a leukocyte interferon-gamma induction assay and IL-18 was immunolocalized in whole human cornea by immunohistochemistry using a specific anti-IL-18 antibody. RESULTS: IL-18 mRNA and bioactive protein was constitutively expressed by human corneal epithelial cells and upregulated by PMA, LPS and poly dI : dC. The constitutive expression of IL-18 protein immunoreactivity was also demonstrated in the epithelial cells of whole human cornea tissue. CONCLUSIONS: This is the first study demonstrating that corneal epithelial cells are capable of producing the IFN-gamma inducing factor IL-18. Increased bioactive corneal IL-18 production can be induced by a number of pro-inflammatory agents and may play an important role in initiating gamma-interferon-mediated inflammatory responses in the cornea.

Cutting edge: C-C chemokine receptor 6 is essential for arrest of a subset of memory T cells on activated dermal microvascular endothelial cells under physiologic flow conditions in vitro.

Memory T cells (mTC) express multiple chemokine receptors (including CCR4 and CCR6) that may potentially be involved in their arrest on inflamed endothelia. Herein, we specifically addressed whether CCR6 is required for mTC to arrest on TNF-alpha-activated human dermal microvascular endothelial cells (HDMEC) in vitro under shear stress conditions. Recombinant liver and activation-regulated chemokine (LARC)/CCL20 (a CCR6 ligand) induced firm arrest of cutaneous lymphocyte Ag(+) mTC in a flow chamber system using purified substrates. Strikingly, desensitization of CCR6 with LARC, but not thymus and activation-regulated chemokine/CCL17 or secondary lymphoid tissue chemokine/CCL21, caused a 50-75% decrease (p < 0. 001) in arrest of mTC on HDMEC, which was indistinguishable from the reduction observed when total mTC were treated with pertussis toxin (p > 0.5). CCR6-depleted mTC also had a markedly reduced ability to arrest on HDMEC. Our results suggest that LARC production by activated endothelial cells and CCR6 expression by mTC may be critical components in the pertussis toxin-sensitive arrest of mTC on activated HDMEC.

Sweet's syndrome in a patient with idiopathic progressive bilateral sensorineural hearing loss.

Acute febrile neutrophilic dermatosis, or Sweet's syndrome, was first described in 1964 and consists of a triad of erythematous cutaneous plaques infiltrated by neutrophils in association with fever and leukocytosis. Sweet's syndrome has been reported to be associated with conditions ranging from upper respiratory tract infections to inflammatory bowel disease to rheumatoid arthritis. We report a patient with a 2-year history of Sweet's syndrome in whom idiopathic progressive bilateral sensory neural hearing loss (IPBSNHL) developed. IPBSNHL is a suspected immunologically mediated hearing loss first described by McCabe in 1979. On the basis of this association, hearing evaluation should be considered in the initial and subsequent examinations of the patient with Sweet's syndrome.

Neural regulation of endothelial cell-mediated inflammation.

There is increasing evidence that the cutaneous neurosensory system can directly modulate inflammatory responses in the skin by the release of neuropeptides such as substance P (SP). Dermal microvascular endothelial cell (DMEC) cellular adhesion molecule (CAM) expression plays a key role in directing leukocyte trafficking during cutaneous inflammatory responses. In recent studies, our laboratory examined the direct effect of SP on DMEC CAM expression and function in vitro and in vivo. Our studies indicate that DMEC express high affinity functional receptors for SP. After exposure to SP, DMEC expressed significant levels of both intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which was accompanied by increased binding to leukocytes expressing the appropriate integrin counter receptors for these CAM. We then determined the in vivo effect of released neuropeptides on DMEC CAM expression. Our results indicate that the topical cutaneous application of the neuropeptide-releasing agent capsaicin resulted in increased ICAM-1 and VCAM-1 immunostaining of microvascular cells in the skin of human volunteers. Little is known regarding the cellular regulatory events by which SP modulates DMEC CAM expression. Our studies indicate that SP-induced cellular Ca+2 signals led to the activation of the NF-kappaB pathway, resulting in nuclear translocation of p65/p50 heterodimers that bind to high-affinity tandem kappaB sites on the VCAM-1 promoter, whereas SP activation induced NF-AT activation and ICAM-1 DNA binding. Thus, these studies further support the role of the cutaneous neurologic system in modulating inflammatory processes in the skin.

Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene.

Interleukin-18 is a potent inducer of interferon-gamma by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-beta-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-gamma expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-gamma-dependent inflammatory processes through expression, activation, and secretion of interleukin-18.

Substance P activates coincident NF-AT- and NF-kappa B-dependent adhesion molecule gene expression in microvascular endothelial cells through intracellular calcium mobilization.

Upon stimulation, cutaneous sensory nerves release neuropeptides such as substance P (SP), which modulate responses in the skin by activating a number of target cells via neurokinin receptors. We have demonstrated that SP preferentially binds to the NK-1R on human dermal microvascular cells, resulting in increased intracellular Ca2+ and induction of ICAM-1 and VCAM-1 expression. In the current studies, we identify specific elements in the regulatory regions of ICAM-1 and VCAM-1 genes as necessary and sufficient for SP-dependent transcriptional activation. SP treatment of human dermal microvascular endothelial cells leads to coincident activation and binding of the transcription factor NF-AT to the -191/-170 region of the ICAM-1 gene (a region bound by activated p65/p65 homodimers in response to TNF-alpha), and NF-kappa B (p65/p50) to tandem NF-kappa B binding sites at -76/-52 of the VCAM-1 gene. The SP-elicited intracellular Ca2+ signal was required for activation and subsequent binding of both NF-AT and NF-kappa B. The transacting factor induction by SP was specific, since a selective NK-1R antagonist blocked SP activation and subsequent NF-AT and NF-kappa B activation and binding. These data demonstrate coincident activation of NF-AT and NF-kappa B via SP-induced intracellular Ca2+ mobilization and indicate a crucial role for neuropeptides in modulating localized cutaneous inflammatory responses.

VCAM-1 expression on human dermal microvascular endothelial cells is directly and specifically up-regulated by substance P.

Sensory nerves in skin are capable of releasing multiple neuropeptides, which modulate inflammatory responses by activating specific cutaneous target cells. Extravasation of particular subsets of leukocytes depends upon the regulated expression of cellular adhesion molecules such as VCAM-1 on microvascular endothelial cells. We examined the direct effect of cutaneous neuropeptides on the expression and function of human dermal microvascular endothelial cell (HDMEC) VCAM-1. A significant increase in VCAM-1 immunostaining of microvascular endothelium was observed in vivo following capsaicin application to human skin. Multiple cutaneous sensory C-fiber-released neuropeptides were evaluated for their ability to induce VCAM-1 cell surface expression on HDMEC. Only substance P (SP) was found to be capable of inducing HDMEC VCAM-1 expression. This SP-mediated VCAM-1 induction appeared to be a direct effect that did not require the release of other HDMEC-derived soluble factors. Increased HDMEC VCAM-1 mRNA expression was detected 1 h after the addition of SP, with peak mRNA increase at 6-9 h postinduction. FACS studies demonstrated a 6.5-fold increase in endothelial cell surface VCAM-1 expression detectable 16 h after addition of SP, which was specifically blocked by a neurokinin-1 receptor antagonist. Increased VCAM-1 cell surface expression on SP-treated HDMEC resulted in a 4-fold increase in the functional binding of 51Cr-labeled MOLT-4 T cells. These data indicate that SP is capable of directly and specifically up-regulating functional endothelial VCAM-1 expression and thus may play a key role in modulating certain inflammatory responses in the skin.

Culture and characterization of sinusoidal endothelial cells isolated from human liver.

Although most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.

Transcriptional activation of the intercellular adhesion molecule 1 (CD54) gene by human T lymphotropic virus types I and II Tax is mediated through a palindromic response element.

In vitro infection of T cells with human T lymphotropic virus types I and II (HTLV-I and HTLV-II) resulted in constitutive expression of ICAM-1. Higher levels of ICAM-1 mRNA were expressed in HTLV-transformed cell lines (MT-2, MoT, C8166) when compared with uninfected T cell lines (A301). We demonstrate that this activation is conferred through a site on the ICAM-1 promoter that is activated in trans by the Tax protein of HTLV-I and HTLV-II. Enhanced promoter activity was detected when the ICAM-1 construct (-1162/+1) was transfected into HTLV-I-infected (MT-2), HTLV-II-infected (MoT, AI 1050), or an HTLV-I Tax-only-expressing (C8166) cell line as compared to the uninfected T cell line (A3.01). Cotransfection of the uninfected T cell line A3.01 with the ICAM construct along with Tax-I and Tax-II expression plasmid also resulted in increased promoter activity. Furthermore, experiments with deletion constructs of the ICAM-1 promoter region indicated that a region between -88 and -53 bp relative to the transcription start site is sufficient for Tax-inducible CAT expression. This segment includes an 11-bp palindromic segment (TTTCCGGGAAA) that has homology with the IFN-gamma and IL-6 response element. An 11-bp segment containing this regulatory region proved to be sufficient to confer Tax-I and Tax-II inducibility on a heterologous promoter (TK-CAT). Taken together these findings indicate that constitutive expression of ICAM-1 by HTLV-infected cells is influenced by the viral trans-activator protein Tax. This increased expression of ICAM-1 in response to the Tax protein may play an important role in the lymphoproliferation associated with HTLV infection.

Ionizing radiation induces, via generation of reactive oxygen intermediates, intercellular adhesion molecule-1 (ICAM-1) gene transcription and NF kappa B-like binding activity in the ICAM-1 transcriptional regulatory region.

Ionizing radiation produces reactive oxygen intermediates in mammalian tissues and may serve as a model system for the investigation of the biologic effects of free radicals. We have previously shown that the adhesion molecule ICAM-1 is induced by ionizing radiation, and here we have investigated the molecular mechanisms responsible. ICAM-1 mRNA and cell surface expression was induced in HeLa and HaCaT cells after exposure to ionizing radiation. This induction was blocked by preincubation with the antioxidants PDTC and N-acetyl cysteine. ICAM-1 promoter activity was assessed by transiently transfecting HeLa cells with CAT-reporter gene constructs containing sequential ICAM-1 5' deletions. ICAM-1 5' fragments -1162/+1 (relative to the transcription start site) and -277/+1 displayed increased promoter activity when cells were exposed to ionizing radiation, but no induction was seen in a -182/+1 construct associating positions -277 to around -182 with inducibility by ionizing radiation. Nuclear extracts from HaCaT cells were tested in mobility shift assays using an NF kappa B-like binding site of the ICAM-1 5' region (positions -186/-177). There was marked enhancement of DNA-protein complex forming in extracts from irradiated versus untreated cells. Incubation of cells with antioxidants prior to irradiation prevented the radiation-dependent increase in complex formation. We conclude that reactive oxygen intermediates are involved in ICAM-1 induction by ionizing radiation. The ionizing radiation-induced, antioxidant-inhibitable binding at the ICAM-1 NF kappa B-like binding site is consistent with the view that NF kappa B is a pro-oxidant transcription factor.

Interactions of the skin and nervous system.

There is increasing experimental evidence that the neurologic system can directly participate in cutaneous inflammation and wound healing. Recent studies indicate that neuropeptides released by cutaneous nerves such as c-fibers can activate a number of target cells including keratinocytes, Langerhans cells, mast cells, and endothelial cells. One such neuropeptide, substance P (SP), is able to specifically bind to murine and human keratinocytes and induce the release of cytokines such as interleukin 1 (IL-1). Other studies demonstrate that SP can also activate mast cells to produce the potent pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha). More recently, we examined the effect of cutaneous neuropeptides on human dermal microvascular endothelial cell (HDMEC) activities. Our studies indicate that the c-fiber-derived calcitonin gene-related peptide (CGRP) is capable of stimulating HDMEC to secrete the neutrophil chemotactic factor interleukin 8 (IL-8). In addition, SP is able to directly activate HDMEC to express high levels of the important cellular adhesion molecule vascular cellular adhesion molecule 1 (VCAM-1). Thus, these studies support the role that the neurologic system may play in mediating the biologic processes that occur during inflammation and wound healing in the skin.

Flanking sequences for the human intercellular adhesion molecule-1 NF-kappaB response element are necessary for tumor necrosis factor alpha-induced gene expression.

The regulated expression of intercellular adhesion molecule-1 (ICAM-1) by cytokines such as tumor necrosis factor alpha (TNF-alpha) plays an important role in inflammation and immune responses. Induction of ICAM-1 gene transcription by TNF-alpha has previously been shown to be dependent upon a region of the ICAM-1 5'-flanking sequences that contains a modified kappaB site. We demonstrate here that this modified kappaB site alone is insufficient for induction of transcription by TNF-alpha. Site-directed mutagenesis of both the kappaB site and specific flanking nucleotides demonstrates that both the specific 5'- and 3'-flanking sequences and the modified kappaB site are necessary for TNF-alpha induction. Further, site-directed mutagenesis of this modified kappaB site to a consensus kappaB site allows it to mediate transcriptional activation in response to TNF-alpha, even in the absence of specific flanking sequences. Transcription through this minimal ICAM-1 TNF-alpha-responsive region can be driven by co-expression of p65, and the minimal response element interacts with p65 and p50 in supershift mobility shift assays. However, when in vitro transcription/translation products for the Rel proteins are used in an electrophoretic mobility shift assay, only p65 is capable of binding the minimal response element while both p50 and p65 bind a consensus kappaB oligonucleotide. Additionally, in the absence of the specific flanking nucleotides, the ICAM-1 kappaB site is incapable of DNA-protein complex formation in both electrophoretic mobility shift assay and UV cross-linking/SDS-polyacrylamide gel electrophoresis analysis. These results demonstrate the requirement for specific flanking sequences surrounding a kappaB binding site for functional transcription factor binding and transactivation and TNF-alpha-mediated induction of ICAM-1.

Pervanadate mimics IFNgamma-mediated induction of ICAM-1 expression via activation of STAT proteins.

Differential expression of intercellular adhesion molecule-1 (ICAM-1) in the epidermis plays a critical role in the regulation of cutaneous inflammation, immunologic reactions, and tissue repair. Transcriptional upregulation of ICAM-1 in response to interferon-gamma (IFNgamma) occurs through a palindromic response element pIgammaRE. pIgammaRE is homologous to IFNgamma-activated sequences, which bind to tyrosine phosphorylated members of the transcription factor family known as signal transducers and activators of transcription (STAT). The importance of tyrosine phosphorylation events in the STAT pathway led us to investigate the effect of the protein tyrosine phosphatase inhibitor, pervanadate, on ICAM-1 expression. We show that treatment of A431 cells and human keratinocytes with pervanadate stimulates protein complex formation on pIgammaRE in a time- and concentration-dependent manner. As demonstrated by mobility supershift assays, the pervanadate-stimulated complex is similar to the IFNgamma-stimulated complex and contains Stat1. Pervanadate treatment also led to an increase in overall protein tyrosine phosphorylation and phosphorylation of Stat1, as well as the subsequent increase in ICAM-1 mRNA and cell surface protein levels. These data show that pervanadate can mimic each step in the IFNgamma-mediated pathway leading to ICAM-1 expression, demonstrate the ability of a pharmacologic agent to bypass the standard cytokine-receptor interaction required for increased ICAM-1 expression, and emphasize the importance of protein tyrosine phosphatases and protein tyrosine kinases in mediating inflammatory responses in the skin.

Retinoic acid inhibits the regulated expression of vascular cell adhesion molecule-1 by cultured dermal microvascular endothelial cells.

The regulated expression of cell adhesion molecules (CAM) on endothelial cells is central to the pathogenesis of various inflammatory processes. Retinoic acid and synthetic derivatives have been demonstrated to exert antiinflammatory effects in cutaneous diseases. To determine modes of retinoid action in the modulation of inflammatory responses, we explored effects of all-trans-retinoic acid (t-RA) on the TNFalpha-induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in cultured human dermal microvascular endothelial cells. Pretreatment with t-RA specifically prevented TNFalpha-induced VCAM-1 expression, but not ICAM-1 and E-selectin induction. t-RA significantly reduced VCAM-1-dependent T cell binding to TNFalpha-treated human dermal microvascular endothelial cells as well. This differential modulation of TNFalpha-induced CAM expression by t-RA was reflected at steady state mRNA levels and in nuclear run-on studies. In transcriptional activation studies, the TNFalpha-mediated activation of the human VCAM-1 promoter was inhibited after t-RA treatment, while the ICAM-1 promoter activation was unaffected, indicating that the selective inhibition of CAM expression is regulated in part at the level of gene transcription. Furthermore, the transcriptional inhibition by t-RA appears to be mediated by its effects upon the activation of NF-kappaB-dependent complex formation. Analysis of protein-DNA binding assays revealed marked inhibition of specific NF-kappaB-dependent binding to the tandem NF-KB sites of the VCAM-1 promoter, but not to the functional NF-kappaB motif of the ICAM-1 promoter. The specific inhibition of cytokine-mediated VCAM-1 gene expression in vitro may provide a potential basis by which retinoids exert their biological effects at sites of inflammation in vivo.

Transforming growth factor-alpha-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP-2-dependent DNA binding and transactivation.

The endothelial cell-specific mitogen vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) represents a central regulator of cutaneous angiogenesis. Increased VPF/VEGF expression has recently been reported in psoriatic skin and healing wounds, both conditions in which transforming growth factor-alpha (TGF alpha) and its ligand, the epidermal growth factor receptor, are markedly up-regulated. Since TGF alpha strongly induces VPF/VEGF synthesis in keratinocytes, TGF alpha-mediated VPF/VEGF expression is likely to play a significant role in the initiation and maintenance of increased vascular hyperpermeability and hyperproliferation in skin biology. The objectives of the present studies were to determine the molecular mechanisms responsible for TGF alpha-induced transcriptional activation of the VPF/VEGF gene. We have identified a GC-rich TGF alpha-responsive region between -88 bp and -65 bp of the VPF/VEGF promoter that is necessary for constitutive and TGF alpha-inducible transcriptional activation. In electrophoretic mobility shift assays, this region binds Sp1-dependent protein complexes constitutively and an additional TGF alpha-inducible protein complex that is distinct from Sp1 protein. Both AP-2 and Egr-1 transcription factors were detected as components of the TGF alpha-inducible protein complex in supershift EMSA studies. In co-transfection studies, an AP-2 but not an Egr-1 expression vector activated VPF/VEGF transcription, thus indicating that AP-2 protein is functionally important in TGF alpha-induced VPF/VEGF gene expression. By clarifying regulatory mechanisms that are critical for angiogenic processes in the skin, these studies may form the basis for new therapeutic strategies to modulate VPF/VEGF expression in cutaneous inflammation and wound healing.

Interferon gamma-dependent induction of human intercellular adhesion molecule-1 gene expression involves activation of a distinct STAT protein complex.

In response to interferon gamma (IFNgamma), intercellular adhesion molecule-1 (ICAM-1) is expressed on human keratinocytes, a cell type that is critically involved in cutaneous inflammation. An ICAM-1 5' regulatory region palindromic response element, pIgammaRE, has been shown to confer IFNgamma-dependent transcription enhancement. By electrophoretic mobility shift assays (EMSA), pIgammaRE forms a distinct complex with proteins from IFNgamma-treated human keratinocytes, termed gamma response factor (GRF). Binding of GRF is tyrosine phosphorylation-dependent, and mutations of pIgammaRE that disrupt the palindromic sequence or alter its spatial relationship abrogate GRF binding. Supershift EMSAs using antibodies to characterized STAT proteins suggest that GRF contains a Stat1alpha-like protein; however, non-ICAM-1 IFNgamma-responsive elements (REs) known to bind Stat1alpha homodimers fail to compete for GRF binding in EMSA, and pIgammaRE does not cross-compete with these REs that complex with homodimeric stat1alpha. The pIgammaRE x GRF complex also displays a distinctly different electrophoretic mobility compared to that of IFNgammaREs complexed to homodimeric Stat1alpha. These findings indicate that a distinct complex containing a Stat1alpha-like protein mediates IFNgamma-induced ICAM-1 gene transcription and identifies a subset of IFNgamma-responsive genes that appear to be regulated by this complex.