Granulocyte-macrophage colony-stimulating factor increases synthesis and expression of CR1 and CR3 by human peripheral blood neutrophils

Polymorphonuclear leukocytes (PMN) constitutively synthesize various plasma membrane proteins including CR1(3) (CD35), CR3 (or Mac-1) alpha-chain (CD11b) and MHC class I. PMN are also able to up-regulate rapidly the expression of CR1 and CR3 to the plasma membrane in response to agonists such as FMLP. To determine whether constitutive PMN translation was static or up-regulatable, PMN were cultured in the presence or absence of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) for 8 h. CR1, CR3 and class I proteins immunoprecipitated from lysates of 35S-methionine pulse-labeled PMN were resolved by SDS-PAGE, fluorographed and quantified by densitometry. GM-CSF-treated PMN synthesized 4.5-fold more class I protein, 3.7-fold more CR1, 2.4-fold more CD11b and 3.4-fold more CR3 beta-chain (CD18), compared with untreated control cells. Actinomycin D treatment of replicate samples of PMN decreased the amount of these proteins synthesized by each group of PMN from 30 to 90%, implying that continued translation was required for the increases in protein synthesis. Nascent CR and class I proteins were inserted into the plasma membrane of PMN, thereby supplementing the molecules already expressed on the cell surface. In addition to these longer term effects of GM-CSF, we observed its acute up-regulatory effects on PMN. GM-CSF induced a five- to 12-fold increase in the expression of CR1 and CR3 on the PMN cell surface within 30 min. These increases were both dose- and time-dependent with maximum up-regulation occurring at 25 pM and at 30 min. In contrast to the long term biosynthetic events, this rapid up-regulation was not dependent on protein synthesis but was due instead to mobilization of CR from intracellular compartments similar to those up-regulated by FMLP. These results demonstrate that PMN can respond to microenvironmental stimuli such as GM-CSF both by rapidly up-regulating and increasing translation and expression of functionally important plasma membrane proteins.     

Expression of carbamoyl-phosphate synthetase I mRNA in Reuber hepatoma H-35 cells. Regulation by glucocorticoid and insulin

Reuber hepatoma H-35 cells actively synthesize the urea cycle enzyme, carbamoyl-phosphate synthetase I. Treatment of H-35 cells with dexamethasone (0.14 microM), however, enhanced synthesis of the enzyme (as measured by incorporation of [35S]methionine) by 4-5-fold. Insulin (0.18 microM) completely inhibited dexamethasone-dependent stimulation of enzyme synthesis. In vitro translation and cDNA hybridization assays were employed to measure effects of dexamethasone plus or minus insulin on levels of mRNA encoding the biosynthetic precursor of carbamoyl-phosphate synthetase I (pCPS) in Reuber H-35 cells. Both measurements yielded similar results: dexamethasone increased pCPS mRNA levels by 4-5-fold and insulin suppressed this response, but only by 50%. Specific cDNA hybridization assays also demonstrated that Reuber H-35 cells, even after hormone treatments, contain only very low levels of albumin mRNA, and no detectable ornithine carbamoyl-transferase mRNA.     

Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation

Murine erythroleukemia (MEL) cells represent a valuable system to study the biogenesis of the cytoskeleton during erythroid differentiation. When attached to fibronectin-coated dishes MEL cells induce, upon addition of DMSO, a 7-d differentiation process during which they enucleate and reach the reticulocyte stage (Patel, V. P., and H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118); they accumulate band 3, spectrin, and ankyrin in amounts equivalent to those found in mature red blood cells. To follow the biosynthesis of spectrin during differentiation, membranes and cytoskeletal proteins of cells metabolically labeled with [35S]methionine were solubilized by SDS and alpha and beta spectrins were recovered by specific immunoadsorption. In both uninduced and 3-d induced cells, the relative synthesis of alpha/beta spectrin is approximately 1:3. In uninduced MEL cells newly synthesized alpha and beta spectrins are degraded with a similar half-life of approximately 10 h. In contrast, in 3-d differentiated MEL cells newly made beta spectrin is much more unstable than alpha spectrin; the half-lives of alpha and beta spectrin chains are approximately 22 and 8 h, respectively. Thus, accumulation of equal amounts of alpha and beta spectrin is caused by unequal synthesis and unequal degradation. As judged by Northern blot analyses, the level of actin mRNA is relatively constant throughout the 7-d differentiation period. alpha and beta spectrin mRNAs are barely detectable in uninduced cells, increase during the first 4 d of induction, and remain constant thereafter. In contrast, band 3 mRNA is first detectable on day 4 of differentiation. Thus, most of the spectrin that accumulates in enucleating reticulocytes is synthesized during the last few days of erythropoiesis, concomitant with the onset of band 3 synthesis. To determine whether this was occurring in normal mouse erythropoiesis, we analyzed the rate of appearance of labeled membrane proteins in mature erythrocytes after a single injection of [35S]methionine. Our results show that most of the spectrin and band 3 in mature erythrocytes is synthesized during the last days of bone marrow erythropoiesis, and that, in the marrow, band 3 and protein 4.1 are synthesized at a somewhat later stage of development than are alpha and beta spectrin, ankyrin, and actin.     

Protein disulphide isomerase from human peripheral blood neutrophils.

Protein disulphide isomerase (PDI) is a 56 kDa resident polypeptide of the endoplasmic reticulum of many cell types. We evaluated the ability of human peripheral blood polymorphonuclear neutrophils (PMN) to synthesize both mRNA and proteins. Using in vitro [35S]-methionine labeling of purified PMN, followed by immunoprecipitation of cell lysates with immobilized polyclonal and monoclonal antibodies and analysis by gel electrophoresis, PMN were shown to synthesize many proteins, including actin. In contrast, incorporation of [35S]-methionine into PDI was not detected. Purification of total RNA from PMN and analysis by Northern blots demonstrated the presence in PMN of PDI-RNA. Western immunoblot evaluations of total PMN protein display an immunoreactive-PDI of 56 kDa. Indirect immunofluorescence studies suggest an abundance of immunoreactive-PDI throughout PMN. We therefore conclude that PDI is synthesized in precursor cells of the bone marrow. Phorbol 12-myristate 13-acetate, a reagent known to affect the degranulation of specific granules, causes the release of immunoreactive-PDI into a post-centrifugation supernatant. PDI, a ubiquitous endoplasmic reticulum resident protein, is shown here to be associated with specific granules in a cell type which has lost its intracellular membrane network during terminal differentiation.     

Regulation of MHC protein expression in pancreatic beta-cells by interferon-gamma and tumor necrosis factor-alpha

Isolated human and mouse pancreatic islet cells and the rat insulinoma cell line RIN-m5F were used to examine the ability of recombinant interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) to regulate the expression of the class I and class II major histocompatibility (MHC) surface proteins and mRNA in beta-cells. Each cytokine increased significantly the expression of class I MHC proteins as determined by double indirect immunofluorescence microscopy and flow cytofluorimetric analysis. In the RIN-m5F cells, this increase in surface expressed class I MHC proteins was mirrored by an increase in the level of class I MHC mRNA. The order of potency of the cytokines on class I MHC expression was TNF-alpha plus IFN-gamma greater than or equal to IFN-gamma greater than or equal to TNF-alpha. While IFN-gamma or TNF-alpha alone were without effect, in combination they were found to induce class II MHC proteins on 30-40% of human or murine beta-cells. In contrast, IFN-gamma plus TNF-alpha did not induce detectable class II MHC proteins or mRNA in the RIN-m5F cells. These findings indicate that 1) TNF-alpha, in addition to IFN-gamma, upregulates the expression of beta-cell class I MHC proteins and mRNA, and 2) more than one signal is required for the induction of class II MHC proteins on beta-cells. The ability of IFN-gamma plus TNF-alpha to induce class II MHC proteins on only a fraction of the normal beta-cell population and not on RIN-m5F cells suggests that this response is related to the differentiation state of the beta-cell.     

Beta-chain broadens range of CD8 recognition for MHC class I molecule.

It is known that the alpha-chain of CD8 binds to a negatively charged loop composed of residues 223 to 229 on MHC class I Ag and that binding of CD8 alpha enhances Ag recognition of T cells. We have recently shown that the mouse CD8 alpha homodimer does not bind to either the HLA class I alpha 3 domain or a mutant of H-2Kb Ag containing a substitution of glutamine for methionine at residue 224, which brings this residue toward the human consensus. Here we report a complementary study of the CD8 beta-chain. The functional role of the CD8 beta-chain was analyzed by using four T cell hybridoma lines expressing mouse CD8 alpha and transfected with the mouse CD8 beta gene. As compared with the lines expressing only CD8 alpha, allorecognition of the chimeric H-2Kb Ag that contains the HLA class I alpha 3 domain was enhanced in lines expressing both CD8 alpha and -beta. This enhancement was blocked by either anti-CD8 mAb or anti-HLA class I alpha 3 domain mAb. In addition, we show that CD8 alpha beta binds the H-2Kb mutant Ag at residue 224. These results suggest that the beta-chain allows the CD8 alpha beta heterodimer to recognize the chimeric H-2Kb Ag. A model for the role of the beta-chain is presented.     

Actin Heterogeneity in primary embryonic culture cells from Drosophila melanogaster.

We have investigated actin heterogeneity in differentiating primary embryonic cell cultures from Drosophila melanogaster. Proteins labeled with [³⁵S]methionine have been separated using O'Farrell two-dimensional gel electrophoresis. Cultures of heterogeneous cell types synthesize at least three major forms of actin, each differing slightly in isoelectric point. We have used a cell separation technique based on differential cell adhesion in the presence of ethylene glycol-bis(β-aminoethyl ether) N,N′-tetraacetate to prepare cultures either highly enriched for, or highly depleted of, myogenic cells. Postfusion myogenic cells synthesize predominantly the most acidic actin form (actin I), while nonmyogenic cells synthesize almost exclusively the two more basic forms (actins II and III). Synthesis of actins at earlier intervals in myogenic cell differentiation in vitro has also been examined. Immediately prior to the onset of myoblast fusion, the synthesis of actin I represents approximately 40% of total actin synthesis. As myogenic cell differentiation progresses this actin form is synthesized at an increasing rate, relative to actins II and III. Drosophila appears to be quite similar to vertebrates with regard to the number of actin species synthesized, as well as to cell and developmental specificity of actin synthesis.     

Regulation of tubulin and actin synthesis and accumulation during Blastocladiella emersonii development

Actin and alpha and beta-tubulin have been identified in Blastocladiella emersonii by two-dimensional gel electrophoresis and Western blotting. The kinetics of synthesis of these proteins were compared by pulse-labeling experiments with [35S]methionine and with the accumulation of their corresponding mRNAs, translated in a cell-free system. Large increases occur in the rates of actin and alpha- and beta-tubulin biosynthesis during sporulation and there is an accumulation of the corresponding mRNAs. In parallel to the increased synthesis, these cytoskeletal proteins accumulate during the late stage of sporulation.     

Diurnal changes in actin mRNA levels and incorporation of35S-methionine into actin in the rat hypothalamus

1. The in vitro incorporation of 35S-methionine into actin and total soluble proteins, as well as the levels of actin mRNA, were studied in the hypothalamus and frontal cerebral cortex of adult male rats killed at six different time intervals during a 24-hr cycle. 2. The specific activity of total soluble proteins after labeled methionine incubations did not vary as a function of time of day in any of the examined brain regions. 3. The incorporation of 35S-methionine into a 43-kDa protein, corresponding to the electrophoretic mobility of actin, varied diurnally in the hypothalamus, exhibiting a maximum at 1200 hr. Such a diurnal variation was not found in frontal cerebral cortex. 4. Similar results were obtained when labeled methionine incorporation into actin was assessed in hypothalamus and cerebral cortex by an immunoprecipitation procedure. 5. An increase in actin hypothalamic mRNA levels, quantitated by dot-blot analysis, was found at 0800, 4 hr in advance to the maximum in 35S-methionine incorporation to actin. 6. The levels of actin mRNA did not vary significantly as a function of time of day in the frontal cerebral cortex.     

High-resolution two-dimensional electrophoretic analysis of acidic,basic and surface proteins of mouse neutrophils

The proteins from murine neutrophils have been examined using isoelectric focusing and non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis as a second dimension. The major protein, actin, dominates the protein profiles and it appears to be one of the few proteins being synthesised rapidly. In the presence of protease inhibitors, neutrophil (a homogeneous, non-dividing cell population) lysates gave extremely reproducible two-dimensional electrophoretic patterns both with Coomassie blue staining (approx. 200 proteins detected) and with fluorography or autoradiography after [³⁵S]methionine biosynthetic labelling (approx. 450 proteins detected between pH 4 and 7). Biosynthetic labelling was more sensitive than protein staining for some components, although the mature neutrophils did not synthesis certain cellular proteins (e.g., granule proteins such as lactoferrin). Surface labelling of neutrophils (as indicated by the absence of ¹²⁵I associated with actin) yielded more than 20 major ¹²⁵I-labelled proteins on high-resolution electrophoretic maps. The major ¹²⁵I-labelled protein (M_r ≈ 90 kdalton) focused at the acidic end of the gels near pH 4.1. This protein could also be detected after [³⁵S]methionine biosynthetic labelling. All of the high molecular weight components focused over a broad pH range (0.2 pH units). At lease one of the surface components appeared to consist of several discrete charge entities.     

Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed.

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.     

Crystal structure of staphylococcal enterotoxin I (SEI) in complex with a human major histocompatibility complex class II molecule

Superantigens are bacterial or viral proteins that elicit massive T cell activation through simultaneous binding to major histocompatibility complex (MHC) class II and T cell receptors. This activation results in uncontrolled release of inflammatory cytokines, causing toxic shock. A remarkable property of superantigens, which distinguishes them from T cell receptors, is their ability to interact with multiple MHC class II alleles independently of MHC-bound peptide. Previous crystallographic studies have shown that staphylococcal and streptococcal superantigens belonging to the zinc family bind to a high affinity site on the class II beta-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the beta-chain is polymorphic and the MHC-bound peptide forms part of the binding interface. To understand how zinc-dependent superantigens recognize MHC, we determined the crystal structure, at 2.0 A resolution, of staphylococcal enterotoxin I bound to the human class II molecule HLA-DR1 bearing a peptide from influenza hemagglutinin. Interactions between the superantigen and DR1 beta-chain are mediated by a zinc ion, and 22% of the buried surface of peptide.MHC is contributed by the peptide. Comparison of the staphylococcal enterotoxin I.peptide.DR1 structure with ones determined previously revealed that zinc-dependent superantigens achieve promiscuous binding to MHC by targeting conservatively substituted residues of the polymorphic beta-chain. Additionally, these superantigens circumvent peptide specificity by engaging MHC-bound peptides at their conformationally conserved N-terminal regions while minimizing sequence-specific interactions with peptide residues to enhance cross-reactivity.     

Differential requirements for cellular cytoskeleton in human macrophage complement receptor- and Fc receptor-mediated phagocytosis.

We investigated the requirement for cellular cytoskeleton in CR- and FcR-mediated phagocytosis by human monocyte-derived macrophages (M phi). Inhibition of actin microfilament (MF) assembly and stability by cytochalasins B and D completely inhibited M phi phagocytosis of sheep E coated with C3b (EC3b), iC3b (EC3bi), and IgG (EIgG) via CR1, CR3, and FcR, respectively. Ligand-binding to either CR or FcR was not effected by cytochalasins. Nocodazole (NOC), which prevents microtubule (MT) polymerization, and taxol, which causes random polymerization of MT inhibited M phi phagocytosis of EC3b(i) but not EIgG. However, the combination of taxol (5 x 10(-4) M) and NOC (2 x 10(-6) M) augmented M phi CR-mediated phagocytosis. In addition, agents known to increase intracellular cGMP augmented phagocytosis of EC3b(i). Conversely, agents that increase intracellular cAMP inhibited CR-mediated phagocytosis. These agents had no effect on FcR-mediated phagocytosis, and did not effect ligand-binding to CR or FcR. PMA markedly enhanced CR- but not FcR-mediated phagocytosis, and augmentation of CR-mediated phagocytosis by PMA was inhibited by both CD and NOC. In contrast, the synthetic diacylglycerol, 1-oleoyl-2-acetoyl-sn-3-glycerol augmented, and inhibitors of protein kinase C inhibited M phi phagocytosis via CR and FcR. These data indicate that for adherently cultured human M phi: 1) binding of ligand-coated E to CR or FcR does not require an intact cytoskeleton; 2) intact actin microfilament are required for phagocytosis via CR and FcR; 3) phagocytosis via CR1 and CR3 but not FcR is dependent on MT assembly; 4) PMA most likely augments CR-mediated phagocytosis through promotion of MT assembly; and 5) PKC activity is involved in the phagocytic signal generated by both CR and FcR.     

Multiple actins in drosophila melanogaster

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.     

Down-regulation of macrophage Ia mRNA expression by interferon (IFN)-alpha and IFN-beta mediated by de novo synthesized protein

We investigated the regulation of class I and class II major histocompatibility complex (MHC) antigen expression of murine peritoneal macrophages (M phi) by interferons (IFNs) at the mRNA level. Enhancement of class I antigen expression by IFNs (IFN-alpha, beta, and gamma), induction of class II antigen expression by IFN-gamma, and inhibition of class II antigen expression by IFN-alpha or IFN-beta all corresponded to steady-state levels of these MHC-specific mRNAs. Cycloheximide (CHX), a protein synthesis inhibitor, was used to elucidate whether IFN regulation of MHC mRNA expression depends on the newly synthesized proteins. CHX concentration was carefully chosen so that M phi viability was not decreased, total protein synthesis was considerably but not completely inhibited, and suppression of surface class II expression was virtually perfect. Under these conditions CHX did not affect the levels of either class I or class II mRNA, but it prevented IFN-beta from interfering with class II mRNA induction by IFN-gamma. These results indicate that the augmentation of induction and/or accumulation of MHC mRNA by IFNs is not dependent on the de novo synthesis of protein, but the down-regulation of class II mRNA level by IFN-beta is mediated by some newly synthesized protein(s).     

Differential interaction of the C3b/C4b receptor and MHC class I with the cytoskeleton of human neutrophils

As measured by fluorescence microscopy and radioligand binding, C3b/C4b receptors (CR1) became attached to the detergent-insoluble cytoskeleton of human neutrophils when receptors were cross-linked by affinity-purified polyclonal F(ab')2 anti-CR1, dimeric C3b, or Fab monoclonal anti-CR1 followed by F(ab')2 goat anti-mouse F(ab')2. CR1 on neutrophils bearing monovalent anti-CR1 was not attached to the cytoskeleton. In contrast, cross-linked CR1 on erythrocytes and cross-linked MHC Class I on neutrophils were not cytoskeleton associated. A possible role for filamentous actin (F-actin) in the binding of cross-linked CR1 to neutrophil cytoskeleton was suggested by three observations. When neutrophils were differentially extracted with either Low Salt-detergent buffer or High Salt-detergent buffer, stained with FITC-phalloidin, and examined by fluorescent flow cytometry, the residual cytoskeletons generated with the former buffer were shown to contain polymerized F-actin, whereas cytoskeletons generated with the latter buffer were found to be depleted of F-actin. In parallel experiments, High Salt-detergent buffer was also found to release cross-linked CR1 from neutrophils. Second, depolymerization of F-actin by DNAse I released half of the cytoskeletal-associated cross-linked CR1. Third, immunoadsorbed neutrophil CR1, but not MHC Class I or erythrocyte CR1, specifically bound soluble 125I-actin. In addition, Fc receptor and CR3, other phagocytic membrane proteins of neutrophils, specifically bound 125I-actin. These data demonstrate that CR1 cross-linked on neutrophils becomes associated with detergent-insoluble cytoskeleton and that this interaction is mediated either directly or indirectly by actin.