Macrophage inducible nitric oxide synthase gene expression is blocked by a benzothiophene derivative with anti-HIV properties.

Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNF alpha-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharide-elicited production of nitrite (NO(-)(2)) by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO(-)(2) production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFN gamma. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNF alpha, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNF alpha synthesis. Additionally, PD144795 did not block NF-kappa B nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug.

Functional differences between monocyte chemotactic protein-1 receptor A and monocyte chemotactic protein-1 receptor B expressed in a Jurkat T cell.

The monocyte chemotactic protein-1 (MCP-1) receptor (MCP-1R) is expressed on monocytes, a subpopulation of memory T lymphocytes, and basophils. Two alternatively spliced forms of MCP-1R, CCR2A and CCR2B, exist and differ only in their carboxyl-terminal tails. To determine whether CCR2A and CCR2B receptors function similarly, Jurkat T cells were stably transfected with plasmids encoding the human CCR2A or CCR2B gene. Nanomolar concentrations of MCP-1 induced chemotaxis in the CCR2B transfectants that express high, intermediate, and low levels of MCP-1R. Peak chemotactic activity was shifted to the right as receptor number decreased. Five-fold more MCP-1 was required to initiate chemotaxis of the CCR2A low transfectant, but the peak of chemotaxis was similar for the CCR2A and CCR2B transfectants expressing similar numbers of receptors. MCP-1-induced chemotaxis was sensitive to pertussis toxin, implying that both CCR2A and CCR2B are G(i)alpha protein coupled. MCP-1 induced a transient Ca(2+) flux in the CCR2B transfectant that was partially sensitive to pertussis toxin. In contrast, MCP-1 did not induce Ca(2+) flux in the CCR2A transfectant. Since MCP-1 can stimulate chemotaxis of the CCR2A transfectant without inducing Ca(2+) mobilization, Ca(2+) flux may not be required for MCP-1-induced chemotaxis in the Jurkat transfectants. These results indicate that functional differences exist between the CCR2A and CCR2B transfectants that can be attributed solely to differences in the carboxyl-terminal tail.

Ribozymes as tools for therapeutic target validation in arthritis.

In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5' untranslated region, the coding region, and the 3' untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.

New reporter cell lines to study macrophage-tropic HIV envelope protein-mediated cell-cell fusion.

The infection of human cells by HIV-1 virus can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 (huCD4) and appropriate human chemokine receptors. In this study, a macrophage-tropic (M-tropic) HIV cell-cell fusion assay was established that utilized huCD4, human CCR5 (huCCR5), and HIV ADAgpl60 as fusion components and a Gal4/VP16-activated luciferase as a reporter system. By combining CHO cells expressing huCD4 and huCCR5 with CHO cells expressing HIV ADAgpl60, a 300-fold increase in luciferase activity could be elicited relative to control. No luciferase activity was detected when HXB2gpl60 (T-tropic) was used instead of ADAgpl60 (M-tropic) as the fusion partner in the assay. Addition of anti-huCD4 (RPA-T4) or anti-huCCR5 (2D7) monoclonal antibodies in the assay significantly inhibited the fusion event; in contrast, an anti-CXCR4 (12G5) monoclonal antibody had little effect, indicating that the fusion assay was huCD4 and huCCR5 dependent. The cell-cell fusion occurred in a time-dependent manner; the maximum luciferase activity was detected about 8 hr after mixing the cells. The fusion events could also be monitored by another reporter system in which Gal4/VP16 activated green fluorescent protein (GFP) was used as the reporter instead of luciferase. In combination with fluorescence microscopy, the GFP reporter system allowed visualization of the fusion events in real time. Compared with previously described HIV fusion models, this system has several advantages, including simplicity, sensitivity, and the ability to allow continuous monitoring of the HIV cell-cell fusion event. Finally, this cell-cell fusion system is easily adapted to study other HIV fusion events.

Use of a beta1 integrin-deficient human T cell to identify beta1 integrin cytoplasmic domain sequences critical for integrin function.

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in beta1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the beta1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human beta1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the beta1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the beta1 integrin with the activating beta1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the beta1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of beta1 integrin structure and function in human T cells.

Identification of a proline-rich sequence in the CD2 cytoplasmic domain critical for regulation of integrin-mediated adhesion and activation of phosphoinositide 3-kinase.

The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in beta1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of beta1 integrin activity. CD2-induced increases in beta1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of beta1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.

Regulation of beta 1-integrin-mediated cell adhesion by the Cbl adaptor protein.

BACKGROUND: Leukocyte activation results in a rapid increase in adhesion to the extracellular matrix due to the activation of beta 1 integrin receptors. A role for phosphatidylinositol (PI) 3-kinase in integrin activation has been proposed, as activation of integrins by many receptors can be blocked by PI 3-kinase inhibitors. One receptor that regulates integrins is the CD28 surface antigen; here, we investigated the mechanisms responsible for CD28-mediated integrin activation. RESULTS: CD28-mediated integrin activation was blocked by mutation of the binding site for the p85 catalytic subunit of PI 3-kinase in the CD28 cytoplasmic domain, and by expression of a dominant-negative form of the p85 subunit. Substitution of the Src homology 2 (SH2)-binding motif in the CD28 cytoplasmic domain for the corresponding motif in the CD28-related CTLA-4 surface antigen also blocked integrin activation but did not affect the recruitment and activation of PI 3-kinase. Mutations of the CD28 cytoplasmic domain that blocked integrin activation also impaired the tyrosine phosphorylation of the Cbl adaptor protein and the activation of the PI 3-kinase that was associated with Cbl. This Cbl-associated PI 3-kinase was distinct from the PI 3-kinase that coprecipitated with the CD28 cytoplasmic domain. CD28-mediated activation of beta 1 integrins was inhibited by expression of a mutant Cbl protein that shows reduced association with PI 3-kinase. CONCLUSIONS: Cbl is required for PI-3-kinase-dependent regulation of integrin receptors by CD28. Furthermore, CD28 is coupled to two distinct pools of PI 3-kinase, one directly associated with the CD28 cytoplasmic tail and the other associated with Cbl.

p53 transactivation of the HIV-1 long terminal repeat is blocked by PD 144795, a calcineurin-inhibitor with anti-HIV properties.

Previous reports have indicated that benzothiophenes exhibit broad anti-inflammatory properties and inhibit human immunodeficiency virus-type 1 (HIV-1) replication. We show that the immunosuppressant cyclosporin A (CsA) and benzothiophene-2-carboxamide, 5-methoxy-3-(1-methyl ethoxy)-1-oxide (PD 144795) block the induction of p53 and NF-kappaB binding to the HIV-1 long terminal repeat (LTR) by the T cell receptor activator phytohemagglutinin. CsA and PD 144795 also inhibit the induction by phytohemagglutinin of the transcription mediated by an HIV-1 LTR fragment containing the p53 and NF-kappaB sites. These effects of PD 144795 on HIV-1 transcription correlate with its ability to inhibit the phosphatase activity of calcineurin and are similar to those previously described for CsA. Moreover, a constitutive active form of calcineurin is able to induce expression from the HIV-1 LTR in a p53- and NF-kappaB-dependent manner and PD 144795 is able to block this induction. These results demonstrate that the DNA binding of p53 to the HIV-1 LTR can be modulated by calcineurin and provide a framework to understand the anti-HIV properties of benzothiophene derivatives.

Structural requirements for beta1 integrin-mediated tyrosine phosphorylation in human T cells.

The beta1 integrin adhesion receptors activate signal transduction pathways that induce tyrosine phosphorylation of a variety of substrates. Increased tyrosine phosphorylation is mediated by the beta1 subunit cytoplasmic domain, which consists of 46 amino acids and contains no intrinsic kinase activity. In the H9 T cell line, beta1 integrin engagement leads to the increased tyrosine phosphorylation of three 105 to 115-kDa substrates that are distinct from focal adhesion kinase (FAK): HEF1 (human enhancer of filamentation 1), a protein with structural homology to p130Cas, and two novel substrates, pp105 and pp115. DNA-mediated gene transfer was used to explore the role of the beta1 cytoplasmic domain in integrin-mediated tyrosine phosphorylation of HEF1, pp105, and pp115 in human T cells. Using a chimeric receptor composed of the cytoplasmic domain of the beta1 integrin subunit and the extracellular and transmembrane domains of the CD2 Ag, we demonstrate that the beta1 cytoplasmic domain is necessary and sufficient for inducing tyrosine phosphorylation of each of these three substrates in H9 T cells. Analysis of a series of beta1 cytoplasmic domain truncations reveals that a truncation of only five amino acids from the carboxyl-terminal end of the beta1 cytoplasmic domain abrogates the ability of the CD2/beta1 chimera to activate tyrosine phosphorylation of HEF1, pp105, or pp115. Thus, the carboxyl-terminal five amino acids, Lys-Tyr-Glu-Gly-Lys (KYEGK), of the beta1 integrin cytoplasmic domain are critical for the coordinate tyrosine phosphorylation of three non-FAK substrates in human T cells.

CD28-mediated up-regulation of beta 1-integrin adhesion involves phosphatidylinositol 3-kinase.

Cross-linking of the CD28 Ag on T cells results in increased beta 1-integrin-mediated adhesion to fibronectin. Chimeric contructs containing the CD28 cytoplasmic domain fused to the extracellular and transmembrane regions of CD2 were expressed in HL60 cells to investigate CD28-mediated regulation of adhesion. Ab cross-linking of the CD2/28 chimera resulted in increased beta 1-dependent adhesion of HL60 transfectants to fibronectin. Induced binding was completely inhibited by the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Cross-linking of the CD2/28 chimera also induced association of the p85 subunit of PI 3-K with the CD2/28 cytoplasmic domain. In contrast, cross-linking of a CD2/28 chimera containing a tyrosine to phenylalanine substitution in the YMNM motif did not result in increased adhesion to fibronectin and did not lead to association of the chimera with PI 3-K. These results directly implicate the YMNM motif and PI 3-K in the regulation of beta 1-integrin activity by the CD28 Ag.

T-lymphocyte interactions with endothelium and extracellular matrix.

T-lymphocyte movement out of the bloodstream and into tissue is critical to the success of these cells in their role in immunosurveillance. This process involves interactions of the T-cell with endothelium as well as with extracellular matrix. Central to these interactions are a number of T-cell adhesion molecules and their endothelial and extracellular matrix ligands. The identification and functional characterization of adhesion molecules have been the subject of intensive research in recent years. We highlight here the latest developments in this rapidly expanding field as they pertain to T-cell interactions with endothelial cells and extracellular matrix components, including: (1) identification of adhesion molecule families, including the selectins, mucins, integrins, immunoglobulin superfamily members, and cadherins; (2) elucidation of the multi-step adhesion cascade that mediates the rolling, arrest, and eventual diapedesis of T-cells through the vascular endothelium into the surrounding tissue; (3) the changes in adhesion molecule expression that accompany T-cell maturation and activation, and the impact of those changes on T-cell migration; (4) the functional relevance of the extracellular matrix for T-cell function; and (5) the clinical relevance of adhesion molecules and the potential for targeting these molecules for the amelioration of immune-mediated diseases.

Nuclease-resistant ribozymes decrease stromelysin mRNA levels in rabbit synovium following exogenous delivery to the knee joint.

Catalytic RNA molecules, or ribozymes, have generated significant interest as potential therapeutic agents for controlling gene expression. Although ribozymes have been shown to work in vitro and in cellular assays, there are no reports that demonstrate the efficacy of synthetic, stabilized ribozymes delivered in vivo. We are currently utilizing the rabbit model of interleukin 1-induced arthritis to assess the localization, stability, and efficacy of exogenous antistromelysin hammerhead ribozymes. The matrix metalloproteinase stromelysin is believed to be a key mediator in arthritic diseases. It seems likely therefore that inhibiting stromelysin would be a valid therapeutic approach for arthritis. We found that following intraarticular administration ribozymes were taken up by cells in the synovial lining, were stable in the synovium, and reduced synovial interleukin 1 alpha-induced stromelysin mRNA. This effect was demonstrated with ribozymes containing various chemical modifications that impart nuclease resistance and that recognize several distinct sites on the message. Catalytically inactive ribozymes were ineffective, thus suggesting a cleavage-mediated mechanism of action. These results suggest that ribozymes may be useful in the treatment of arthritic diseases characterized by dysregulation of metalloproteinase expression.

A comparison of anesthetic techniques for awake intubation in neurosurgical patients.

Two different methods of achieving upper airway anesthesia for awake fiberoptic intubation were prospectively compared in patients undergoing surgery for cervical spine instability. Forty patients were randomized to either topical anesthesia or nerve block groups. Topical anesthesia patients were administered nebulized 4% lidocaine (approximately 20 ml) via the oropharynx plus a transtracheal injection of 4% lidocaine (3 ml). Nerve block patients underwent bilateral glossopharyngeal and superior laryngeal nerve blocks with 2% lidocaine (0.5-2 ml per injection site) plus a transtracheal injection of 4% lidocaine (3 ml). The quality of anesthesia for intubation was graded by observers blinded to group assignment. Mean arterial pressure, heart rate, Pao2, Paco2, pHa, SpO2, and plasma lidocaine concentrations were measured during the intubation sequence. Patient recall of intubation and discomfort were assessed during the postoperative period with visual analog scales. Time required for successful intubation and quality of intubation were not different between groups. Physiologic values for the two groups were similar. The mean total dose of lidocaine in the topical anesthesia group was approximately 2 times greater than that in the nerve block group (815 versus 349 mg; p < 0.0001). In contrast, mean plasma lidocaine concentration at initiation of intubation in the topical anesthesia group was half that of nerve block group (2.16 versus 4.23 micrograms/ml; p < 0.0001). Ten minutes later there was no difference for plasma lidocaine concentration between groups. No patients had evidence of seizures or neurologic change during the procedure. There was no difference in patient perception of discomfort during the procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunodominant autoepitopes of type VII collagen are short, paired peptide sequences within the fibronectin type III homology region of the noncollagenous (NC1) domain.

Autoantibodies to type VII collagen are associated with the blistering diseases epidermolysis bullosa acquisita and bullous systemic lupus erythematosus. We showed previously that these autoantibodies recognize epitopes within the noncollagenous (NC1) region of type VII collagen. That region is composed of fibronectin type III homology units that may contribute to intermolecular cross-linking and basement membrane adhesion functions of type VII collagen. In this study, we defined the specific amino acid sequences recognized by these autoantibodies. By fusion protein analysis, sera from patients with epidermolysis bullosa acquisita and bullous lupus were found to react with two regions within the fourth (E-1) and eighth (E-2) fibronectin homology repeats, each consisting of approximately 100 amino acids. Affinity purification studies showed E-1 and E-2 to be independent and non-cross-reactive epitope regions. These regions were probed further by enzyme-linked immunosorbent assay analysis of overlapping octapeptide sets derived from the amino acid sequences of E-1 and E-2. The results showed two reactive, closely associated octapeptide sequences within each region, both lying in amphipathic portions of fibronectin type III homology repeats. These studies identify short peptide sequences within the NC1 domain of type VII collagen that are targeted independently by autoantibodies. These sequences may play a direct role in determining the properties of type VII collagen that influence adhesion between this molecule and other basement membrane proteins, and their alteration by antibody binding may be the immunopathogenic event underlying epidermolysis bullosa acquisita and bullous lupus.

Autoantibodies to type VII collagen recognize epitopes in a fibronectin-like region of the noncollagenous (NC1) domain.

Autoantibodies to type VII collagen are characteristic of the blistering diseases epidermolysis bullosa acquisita and bullous systemic lupus erythematosus (SLE). Blisters in those diseases are due to defective adhesion of the lamina densa subregion of the epithelial basement membrane to the underlying dermis. Previous studies indicating that type VII collagen contributes to lamina densa-dermal adhesion by cross-linking lamina densa and dermal matrix proteins suggests that autoantibodies may contribute to blisters by interfering with type VII collagen function. That hypothesis is supported by previous studies showing autoantibodies from a small number of epidermolysis bullosa acquisita patients recognize proteolytic fragments containing the 145-kD noncollagenous domain of type VII collagen. In this study, we examined reactivity of autoantibodies from a large number of epidermolysis bullosa acquisita and bullous SLE patients with fusion proteins representing most of the noncollagenous domain of type VII collagen and that those regions are homologous to type III repeats of fibronectin. These results suggest autoantibodies binding to fibronectin homology regions within the 145-kD noncollagenous domain may interfere with the adhesion function of type VII collagen and contribute to lamina densa-dermal dysadhesion in epidermolysis bullous acquisita and bullous SLE.

Techniques for cloning cDNAs encoding interactive transcriptional regulatory proteins.

Several approaches aimed at detecting and cloning interactive transcriptional regulatory proteins have been presented. All of the techniques can effectively identify specific interactions between two transcription proteins. However, interaction cloning and the two hybrid system have the added advantage of yielding a cDNA expression clone directly. The other methods, EMSA-mediated cloning, co-immunoprecipitation, oligonucleotide/PCR-facilitated cloning, Southwestern, and Farwestern, require additional manipulations to obtain a cDNA clone. Clearly, the interactive cloning system of choice will depend on the proteins under investigation.

Noncollagenous (NC1) domain of collagen VII resembles multidomain adhesion proteins involved in tissue-specific organization of extracellular matrix.

Type VII collagen (C7) is a stratified squamous epithelial basement membrane protein composed of three identical alpha chains, each consisting of a 145-kDa amino-terminal noncollagenous (NC1) domain and a 145-kDa carboxyl-terminal collagenous domain. Morphologic and biochemical studies have shown that tissue-specific aggregates of C7 dimers called anchoring fibrils may contribute to epithelial basement membrane organization and adherence by interacting with extracellular matrix (ECM) proteins such as type IV collagen. In this study, we cloned a cDNA encoding most of the NC1 domain of C7. The deduced amino acid sequence revealed motifs characteristic of multidomain ECM proteins that contribute to the tissue-specific organization of ECM including a region of 7 1/2 sequential fibronectin type III (Fn III) homology repeats, a potential collagen-binding region homologous to the A domain of von Willebrand factor (vWf) and an RGD sequence. A purified C7 fusion protein containing these motifs specifically bound to type IV collagen in a functional assay. These results suggest that regions within the NC1 domain of C7 mediate interactions with lamina densa and dermal ECM proteins including type IV collagen. Structural mutations and autoepitopes in these regions may represent mechanisms for the development of defective basement membrane organization and adherence in genetic and autoimmune forms of epidermolysis bullosa.