The receptor for interferon-gamma on human peripheral blood monocytes consists of multiple distinct subunits.

The interaction of interferon-gamma (IFN gamma) (a product of activated T lymphocytes) and monocytes is essential for immune responsiveness, host defense, and chronic inflammation. In this report we define the IFN gamma receptor (IFN gamma R) on human monocytes as a receptor complex consisting of at least three subunits. Solubilization and immunoprecipitation of [35S]methionine- and [35S]cysteine-labeled monocytes were optimized by controlling the detergent concentration during solubilization and washing of the immunoprecipitates. This enabled subunits to be coimmunoprecipitated by several different anti-IFN gamma R antibodies raised against the 90-kDa cloned binding protein. Immunoprecipitation under stringent (1% sodium dodecyl sulfate) conditions resulted in the visualization of only the 80-90-kDa binding protein. Under less stringent conditions at least two coimmunoprecipitated subunits (molecular mass of 200 and 38 kDa) were consistently associated with the 80-kDa (90-92 kDa reduced) binding protein. The 38-kDa subunit was shown to be distinct from the 80-kDa subunit by proteolytic fragment analysis. Cross-linking of 125I-rIFN gamma to monocytes yielded receptor-IFN gamma complexes consistent with the existence of multiple subunits.     

Recombinant 70-kDa protein from the amino-terminal region of rat fibronectin inhibits binding of fibronectin to cells and bacteria

Binding of fibronectin to substrate-attached cells and to Staphylococcus aureus is mediated by the amino-terminal 70-kDa portion of fibronectin. The 70-kDa amino-terminus is composed of nine type I and two type II internal homology units, each containing two intrachain disulfide bonds. The exact structural features of the 70-kDa amino-terminus that are necessary for binding to cells and bacteria are not known. We characterized a recombinant 70-kDa protein from the amino-terminus of rat fibronectin using a baculovirus expression system. Recombinant 70-kDa (r70kDa) protein was easily purified in high amounts from the conditioned medium by affinity chromatography on gelatin-agarose. Secretion was much less when N-linked glycosylation was blocked by tunicamycin. Like the native fragment, the r70kDa protein contains intrachain disulfide bonds. In addition, the r70kDa protein was indistinguishable from the nonrecombinant 70-kDa fragment in its ability to compete for binding sites on fibroblasts and S. aureus. Thus, the r70kDa protein retains the important functional characteristics of the native fragment. This expression system is well adapted to studying the structural features important for the interaction of 70-kDa protein with cells.     

A chemokine,interferon (IFN)-gamma-inducible protein 10 kDa,is stimulated by IFN-tau and recruits immune cells in the ovine endometrium

Proper distribution of immune cells in the uterus is a prerequisite for successful implantation and subsequent placentation, but biochemical signals that govern such events have not been well characterized. In the present study, the cDNA of a chemokine, interferon (IFN)-gamma-inducible protein 10 kDa (IP-10), was identified from a cDNA subtraction study between uterine endometrial tissues from Day 17 pregnant and Day 15 cyclic ewes. The effect of IFN-tau on IP-10 expression and the involvement of IP-10 in the recruitment of immune cells were then investigated. Northern blot analysis revealed that large amounts of IP-10 mRNA were present during conceptus attachment to maternal endometrium and early placentation. IP-10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. This finding was supported by the discovery of IP-10 mRNA expression in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. Moreover, the expression of IP-10 mRNA by the monocytes was stimulated by IFN-alpha, IFN-gamma, and IFN-tau in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-tau. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-tau stimulated-endometrial culture medium, and the effect was significantly reduced by neutralization with an anti-IP-10 antibody. These results suggest that endometrial IP-10 regulated by conceptus IFN-tau regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.     

Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal.

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 X 10(-8) mol of free sulfhydryls and 3.24 X 10(-8) mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Nonreduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin.     

Expression and purification of stable 33-kDa soluble human CD23 using the Drosophila S2 expression system.

CD23, a 45-kDa type II membrane glycoprotein present on B cells, monocytes, and other human immune cells, is a low-affinity receptor for IgE. The extracellular region of the membrane-bound human CD23 is processed into at least four soluble (s) CD23 forms, with apparent molecular masses of 37, 33, 29, and 25 kDa. High levels of sCD23 are found in patients with allergy, certain autoimmune diseases, or chronic lymphocytic leukemia. Therefore, inhibition of the processing of membrane-bound CD23 to control the cytokine-like effects of sCD23 offers a novel therapeutic opportunity. While the 37-, 29-, and 25-kDa forms of sCD23 have been expressed previously as recombinant proteins, the 33-kDa form has not been purified and characterized. To further investigate the multiple roles of sCD23 fragments and to devise assays to identify potent small-molecule inhibitors of CD23 processing, we have produced the 33-kDa form of sCD23 using Chinese hamster ovary (CHO) and Drosophila S2 cells. The CHO-expressed 33-kDa protein was found to undergo proteolytic degradation during cell growth and during storage of purified protein, resulting in accumulation of a 25-kDa form. The Drosophila system expressed the 33-kDa sCD23 in a stable form that was purified and demonstrated to be more active than the CHO-derived 25-kDa form in a monocyte TNFalpha release assay.     

Cycloheximide induces expression of the human interferon beta 1 gene in mouse cells transformed by bovine papillomavirus-interferon beta 1 recombinants.

Mouse cells transformed by a bovine papillomavirus recombinant vector containing the human interferon (IFN) beta 1 (IFN-beta 1) gene could be induced to produce human as well as mouse IFNs. The optimal conditions for induction of human IFN and of its mRNA in these transformants resembled those needed for mouse IFN: high concentrations of DEAE-dextran and low concentrations of polyriboinosinic acid-polyribocytidylic acid. Superinduction by inhibitors of protein synthesis which strongly stimulate IFN-beta 1 induction in human cells had only a small effect on human IFN induction in bovine papillomavirus IFN-beta 1-transformed mouse cells. In contrast, cycloheximide without double-stranded RNA could induce significant levels of human IFN in the bovine papillomavirus IFN-beta 1 mouse transformants. After cycloheximide treatment, these cells contained IFN-beta 1 mRNA whose 5' ends originated in the authentic start site of the human IFN-beta 1 gene, as shown by S1 nuclease mapping. The transferred human gene, propagated extrachromosomally in the mouse cells, was, therefore, inducible under conditions different from those in human cells. The results also confirmed that the inhibitor of protein synthesis, cycloheximide, can induce expression of a human IFN gene.     

Loss of one asparagine-linked oligosaccharide from human transferrin receptors results in specific cleavage and association with the endoplasmic reticulum.

The coding sequence for the human transferrin receptor gene has been mutated in order to abolish the attachment of the oligosaccharide closest to the transmembrane sequence. Expression of the mutant receptor in Chinese hamster ovary cells resulted, after analysis under nonreducing conditions, in an 85- and a 73-kDa receptor species. After reduction, the 85-kDa receptors were mostly converted to 73 kDa, although with short labeling periods some 85-kDa receptor remained suggesting that the mutated gene was capable of coding for the entire polypeptide which was then proteolytically processed. This was supported by in vitro translation of mRNA from either wild type or mutant cells which in both cases yielded an 80-kDa protein, the full size of the nonglycosylated protein. The solubility characteristics of the mutant receptor suggest that it contains the COOH-terminal extracellular domain but not the transmembrane sequence. This is supported by the endoglycosidase H sensitivity of the 73-kDa protein which is compatible with the retention of two of the original three high mannose oligosaccharides. The receptor that lacks one oligosaccharide is unable to form intermonomer disulfide bridges or to migrate to the cell surface and is located in the endoplasmic reticulum where it is further degraded after a lag period of about 30 min.     

Extrinsic 33-kilodalton protein of spinach oxygen-evolving complexes: kinetic studies of folding and disulfide reduction

The 33-kDa protein is one of the three extrinsic proteins in the oxygen-evolving photosystem II complexes. The protein has one intrachain disulfide bond. On reduction of this disulfide bond, the protein was unfolded and lost its activity. On the basis of the unfolding equilibrium curve obtained by using guanidine hydrochloride, the free energy change of unfolding in the absence of guanidine hydrochloride was estimated to be 4.4 kcal/mol using the Tanford method [Tanford, C. (1970) Adv. Protein Chem. 24, 1-95] and 2.8 kcal/mol using the linear extrapolation method. The unfolding of the 33-kDa protein caused by reduction was explained in terms of the entropy change associated with reduction of the intrachain disulfide bond. The kinetics of the reduction of the disulfide bond using dithiothreitol were studied at various concentrations of guanidine hydrochloride at pH 7.5 and 25 degrees C. The disulfide bond was reduced even in the absence of guanidine hydrochloride. The unfolding and refolding kinetics of the 33-kDa protein using guanidine hydrochloride were also studied under the same conditions, and the results were compared with those for the reduction kinetics. It was shown that the reduction of the disulfide bond proceeds through a species in which the disulfide bond is exposed by local fluctuations.     

Heat and sodium arsenite act synergistically on the induction of heat shock gene expression in Xenopus laevis A6 cells

Heat shock protein (HSP) synthesis was studied in the Xenopus epithelial cell line A6 in response to heat and sodium arsenite, either singly or together. Temperatures of 33-35 degrees C consistently brought about the synthesis of HSPs at 87, 73, 70, 54, 31, and 30 kilodaltons (kDa), whereas sodium arsenite at 25-100 microM induced the synthesis of HSPs at 73 and 70 kDa. In cultures exposed to 10 microM sodium arsenite at 30 degrees C, HSP synthesis in the 68- to 73-kDa and 29- to 31-kDa regions was much greater than the HSP synthesis in response to each treatment individually. RNA dot blot analysis using homologous genomic subclones revealed that heat shock induced the accumulation of HSP 70 and 30 mRNAs. The sizes of the HSP 70 and 30 mRNAs determined by Northern hybridization were 2.7 and 1.5 kilobases, respectively. Sodium arsenite (10-100 microM) also induced the accumulation of both HSP 70 and 30 mRNAs. Finally, a mild heat shock (30 degrees C) plus a low concentration of sodium arsenite (10 microM) acted synergistically on HSP 70 and 30 mRNA accumulation in A6 cells. Thus sodium arsenite and heat act synergistically at the level of both HSP synthesis and HSP mRNA accumulation.     

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.     

Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.     

Structural analysis of the sequence coding for an inducible 26-kDa protein in human fibroblasts

When human fibroblast cells were stimulated with poly(I) X poly(C) in the presence of cycloheximide for the production of interferon-beta (IFN-beta), a 26-kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN-beta [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768-2772]. In our hands this 26-kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a so-called beta 2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152-7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26-kDa product. In this paper we describe the nucleotide sequence of the 26-kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26-kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26-kDa protein can be explained. It turns out that the 26-kDa protein gene and the so-called 'IFN-beta 2' gene are identical. However, extensive homology searches indicate that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26-kDa product represents a novel IFN species remains open.     

CXCR3 expression on CD34(+) hemopoietic progenitors induced by granulocyte-macrophage colony-stimulating factor: II. Signaling pathways involved

CXCR3, known to have four ligands (IFN-gamma inducible protein 10 (gamma IP-10), monokine induced by IFN-gamma (Mig), I-TAC, and 6Ckine), is predominantly expressed on memory/activated T lymphocytes. We recently reported that GM-CSF induces CXCR3 expression on CD34(+) hemopoietic progenitors, in which gamma IP-10 and Mig induce chemotaxis and adhesion. Here we further report that stimulation with GM-CSF causes phosphorylation of Syk protein kinase, but neither Casitas B-lineage lymphoma (Cbl) nor Cbl-b in CD34(+) hemopoietic progenitors can be blocked by anti-CD116 mAb. Specific Syk blocking generated by PNA antisense completely inhibits GM-CSF-induced CXCR3 expression in CD34(+) progenitors at both mRNA and protein as well as at functional levels (chemotaxis and adhesion). Cbl and Cbl-b blocking have no such effects. Thus, GM-CSF binds to its receptor CD116, and consequently activates Syk phosphorylation, which leads to induce CXCR3 expression. gamma IP-10 and Mig can induce Syk, Cbl, and Cbl-b phosphorylation in CD34(+) progenitors by means of CXCR3. gamma IP-10 or Mig has induced neither chemotaxis nor adhesion in GM-CSF-stimulated Cbl-b-blocked CD34(+) hemopoietic progenitors, whereas SDF-1alpha induces both chemotaxis and adhesion in these cells. Interestingly, gamma IP-10 and Mig can induce chemotaxis and adhesion in GM-CSF-stimulated Syk- or Cbl-blocked CD34(+) hemopoietic progenitors. Thus, Cbl-b, but not Syk and Cbl phosphorylation, is essential for gamma IP-10- and Mig-induced chemotaxis and adhesion in CD34(+) hemopoietic progenitors. This study provides a useful insight into novel signaling transduction pathways of the functions of CXCR3/gamma IP-10 and Mig, which may be especially important in the cytokine/chemokine environment for mobilization, homing, and recruitment during proliferation, differentiation, and maturation of hemopoietic progenitor cells.     

Interferon gamma bound to endothelial cells is phosphorylated by ecto-protein kinases

The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [gamma-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFN gamma bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFN gamma it was associated with the appearance of a strongly phosphorylated band of 17 kDa corresponding to IFN gamma itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR--a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function.