A soluble form of the murine common gamma chain is present at high concentrations in vivo and suppresses cytokine signaling

The common gamma-chain (c) is a component of the receptors forIL-2, IL-4, IL-7, IL-9, and IL-15 and is essential for their signaltransduction. Western blotting and a newly established enzyme-linkedimmunosorbent assay detected substantial constitutive levels (50-250 ng/mL) of soluble c (sc) in sera of murine inbred strains. It wasdemonstrated that purified immune cells, such as T, B, and naturalkiller cells, and macrophages released this protein after activation.Transfection experiments with cDNA encoding the full-length c showedthat shedding of the transmembrane receptor led to the release ofsc. The shedding enzymes, however, appeared to be distinct fromthose cleaving other cytokine receptors because inhibitors ofmetalloproteases (eg, TAPI) did not influence sc release. In vivo,superantigen-induced stimulation of T cells enhanced sc serumconcentrations up to 10-fold within 6 hours. Because these findingsdemonstrated regulated expression of a yet unknown molecule in theimmune response, further experiments were performed to assess thepossible function(s) of sc. A physiological role of sc wasindicated by its capacity to specifically inhibit cell growth inducedby c-dependent cytokines. Mutational analysis revealed that theC-terminus and the WSKWS motif are essential for the cytokineinhibitory effect of the sc and for binding of the molecule tocytokine receptor-expressing cells. Thus, competitive displacement ofthe transmembrane c by excess sc is the most likely mechanism ofcell growth inhibition. It was implied that naturally produced sc isa negative modulator of c-dependent cytokines.     

Cloning and Characterization of the Human Interleukin-3 (IL-3)/IL-5/ Granulocyte-Macrophage Colony-Stimulating Factor Receptor beta c Gene: Regulation by Ets Family Members

High-affinity receptors for interleukin-3 (IL-3), IL-5, andgranulocyte-macrophage colony-stimulating factor (GM-CSF) are composedof two distinct subunits, a ligand-specific chain and a common  chain (c). Whereas the mouse has two homologous subunits (cand IL-3), in humans, only a single  chain is identified. Wedescribe here the isolation and characterization of the gene encodingthe human IL-3/IL-5/GM-CSF receptor  subunit. The gene spans about25 kb and is divided into 14 exons, a structure very similar to that ofthe murine c/IL-3 genes. Surprisingly, we also found the remnantsof a second c chain gene directly downstream of c. We identifieda functional promoter that is active in the myeloid cell lines U937 andHL-60, but not in HeLa cells. The proximal promoter region, locatedfrom 103 to +33 bp, contains two GGAA consensus binding sites formembers of the Ets family. Single mutation of those sites reducespromoter activity by 70% to 90%. The 5 element specificallybinds PU.1, whereas the 3 element binds a yet-unidentifiedprotein. These findings, together with the observation thatcotransfection of PU.1 and other Ets family members enhances cpromoter activity in fibroblasts, reinforce the notion that GGAAelements play an important role in myeloid-specific gene regulation.     

Presentation of ovalbumin internalized via the immunoglobulin-A Fc receptor is enhanced through Fc receptor gamma-chain signaling

The mechanism of enhanced presentation of ovalbumin (OVA)internalized as immunoglobulin A (IgA)-OVA via the IgA Fc receptor (FcR) was analyzed by focusing on the role of the FcR-associated  chain. Comparison of B-cell transfectants expressing FcR plus wild-type (WT)  chain or  chain in which theimmunoreceptor tyrosine-based activation motif (ITAM) was altered bytyrosine mutation or substitution with the ITAM of FcRIIA showedthat signaling-competent ITAM was not required for endocytosis ofIgA-OVA. However, antigen presentation was impaired by ITAM changes.Signaling-competent -chain ITAM appeared necessary for transport ofligated FcR to a lamp-1⁺ late endocytic compartment forremodeling and/or activation of that compartment and also for efficientdegradation of IgA complexes. Moreover, FcR ligation also activatedefficient processing of nonreceptor-targeted antigen. Theresults suggest that -chain signaling activates the antigenprocessing compartment.     

Coinduction of Embryonic and Adult-Type Globin mRNAs by Sodium Butyrate and Trichostatin A in Two Murine Interleukin-3-Dependent Bone Marrow-Derived Cell Lines

Using an RNase protection assay, globin mRNA species expressed inclones derived from Ba/F3 and B6SUtA cells transfected with theerythropoietin receptor (EpoR) and selected with erythropoietin (Epo)were compared with globin mRNA species induced in corresponding parental cells by sodium butyrate (SB) and trichostatin A (TSA). ^Major/^minor- and -1/-2-globinmRNAs were the major species, with trace amounts of -globin mRNA,formed in Epo-stimulated EpoR⁺ Ba/F3 clones, whereas SBand TSA allowed expression of all species of globin mRNAs, ie, ,h1, ^major/^minor, , and -1/-2,in parental Ba/F3 cells. In contrast, - and -1/-2-globinmRNAs were the major species present in Epo-stimulated EpoR⁺ B6SUtA clones, whereas SB and TSA activated -,h1-, ^S/^T-, and -1/-2-globingenes in parental B6SUtA cells; -globin mRNA was not detected in SB-and TSA-treated B6SUtA cells. Because TSA is a specific inhibitor ofhistone deacetylase, the mimicry of action exhibited by SB and TSAsuggests that the effects of SB are mediated through its ability toinhibit histone deacetylase and that histone deacetylase is an integralpart of the repression of globin genes in theseinterleukin-3-dependent cells. Efficient coinduction of embryonic andadult types of globin mRNA in bone marrow cell lines derived from adultmice indicates that adult hematopoietic precursors possess an embryonicnature. These cell lines are useful models to study the mechanism(s) ofdevelopmental globin gene switching.     

Perforin and interferon-gamma activities independently control tumor initiation, growth, and metastasis

Perforin (pfp) and interferon- (IFN-) together inC57BL/6 (B6) and BALB/c mouse strains provided optimal protection in 3 separate tumor models controlled by innate immunity. Using experimental (B6, RM-1 prostate carcinoma) and spontaneous (BALB/c, DA3 mammary carcinoma) models of metastatic cancer, mice deficient in both pfp andIFN- were significantly less proficient than pfp- or IFN--deficient mice in preventing metastasis of tumor cells to thelung. Pfp and IFN--deficient mice were as susceptible as micedepleted of natural killer (NK) cells in both tumor metastasis models,and IFN- appeared to play an early role in protection frommetastasis. Previous experiments in a model of fibrosarcoma induced bythe chemical carcinogen methylcholanthrene indicated an important rolefor NK1.1⁺ T cells. Herein, both pfp and IFN- playedcritical and independent roles in providing the host with protectionequivalent to that mediated by NK1.1⁺ T cells. Furtheranalysis demonstrated that IFN-, but not pfp, controlled the growthrate of sarcomas arising in these mice. Thus, this is the first studyto demonstrate that host IFN- and direct cytotoxicity mediated bycytotoxic lymphocytes expressing pfp independently contribute antitumoreffector functions that together control the initiation, growth, andspread of tumors in mice.     

Functional Analysis of Mature Hematopoietic Cells From Mice Lacking the beta c Chain of the Granulocyte-Macrophage Colony-Stimulating Factor Receptor

Mice with a null mutation of the c chain of thegranulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin-3 (IL-3), and IL-5 receptors (c-null mice) develop analveolar proteinosis-like lung disease. The pathogenesis of thisdisease is uncertain and, although a defect in alveolar macrophagefunction has been postulated, no previous analysis of maturehematopoietic cells in mice with alveolar proteinosis has beenreported. Therefore, we undertook a functional analysis of the maturehematopoietic cell compartment in c-null mice. In addition, wereexamined the roles of the GM-CSF receptor chain and the cchain in signaling by GM-CSF. Neutrophils and macrophages fromc-null mice were capable of normal survival and phagocytosis in theabsence of stimulus and of similar levels of nitric oxide production inresponse to interferon- and lipopolysaccharide. GM-CSF-mediatedaugmentation of survival, phagocytosis, and hydrogen-ion productionwere absent in neutrophils from c-null mice. Interestingly, we wereunable to show any ability of the GM-CSF receptor -chain alone tomediate glucose transport in these cells. In keeping with the c-null mice lung pathology, examination of lavage fluid from the lungs ofc-null mice showed increased cellularity. This was caused by anincrease in the number of lymphocytes, neutrophils, and macrophages.Large foamy cells in the lavage fluid from c-null mice wereidentified as macrophages using immunohistochemistry. Functionalanalysis showed that these c-null alveolar macrophages were capableof phagocytosis but uptake of colloidal carbon and cellular adhesionwere reduced. In summary, mature hematopoietic cells with a nullmutation of the c receptor were unable to perform GM-CSF-mediatedhematopoietic cell functions including glucose transport, but respondednormally to a range of other ligands.     

Association Between Ligand-Induced Conformational Changes of Integrin alpha IIbbeta 3 and alpha IIbbeta 3-Mediated Intracellular Ca2+ Signaling

Platelet _IIb3 is a prototypic integrinand plays a critical role in platelet aggregation. Occupancy of_IIb3 with multivalent RGD ligands, suchas fibrinogen, induces both expression of ligand-induced binding sites(LIBS) and _IIb3 clustering, which arethought to be necessary for outside-in signaling. However, theassociation between LIBS expression and outside-in signaling remainselusive. In this study, we used various_IIb3-specific peptidomimetic compounds asa monovalent ligand instead of fibrinogen and examined the associationbetween LIBS expression and outside-in signaling such as_IIb3-mediated intracellularCa²⁺ signaling. Using a set of monoclonal antibodies(MoAbs) against LIBS, we showed that antagonists can be divided intotwo groups. In group I, antagonists can induce LIBS on both_IIb and ₃ subunits. In group II,antagonists can induce LIBS on the _IIb subunit, but noton the ₃ subunit. Inhibition studies suggested thatgroup I and group II antagonists interact with distinct but mutually exclusive sites on _IIb3. Neither group Inor group II antagonist increased intracellular Ca²⁺concentrations ([Ca²⁺]_i) in nonactivatedplatelets. All antagonists at nanomolar concentrations abolished theincrease in [Ca²⁺]_i in 0.03 U/mLthrombin-stimulated platelets, which is dependent on bothfibrinogen-binding to _IIb3 andplatelet-aggregation. However, only group I antagonists at higherconcentrations dose-dependently augmented the[Ca²⁺]_i increase, which is due toaggregation-independent thromboxane A₂ production. Thisincrease in [Ca²⁺]_i was not observed inthrombasthenic platelets, which express no detectable_IIb3. Thus, only the group I antagonists,albeit a monovalent ligand, can initiate_IIb3-mediated intracellular Ca²⁺ signaling in the presence of thrombin stimulation.Our findings strongly suggest the association between ₃LIBS expression and _IIb3-mediatedintracellular Ca²⁺ signaling in platelets.     

Ligand binding to integrin alpha(v)beta(3) requires tyrosine 178 in the alpha(v) subunit

Integrin _v3 has been implicatedin angiogenesis and other biological processes. However, theligand-binding sites in _v, a non-I-domain  subunit,remain to be identified. Recently in _IIb, the otherpartner of the ₃ subunit, several discontinuous residuesimportant for ligand binding were identified in the predicted loopsbetween repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alanine-scanning mutagenesis in293 cells was used to investigate the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of _v in ligandbinding. Wild-type _v3 was able to bindsoluble fibrinogen following integrin activation either by 0.5 mMmanganese dichloride (MnCl₂) or a mutation of₃ threonine (T)562 to asparagine. However, mutation oftyrosine (Y)178 to alanine in the predicted G172-G181 loop of_v abolished fibrinogen binding, and alanine (A)substitutions at adjacent residues phenylalanine (F)177 and tryptophan(W)179 had a similar effect. Cells expressing Y178A_valso failed to bind to immobilized fibrinogen. Moreover, the Y178Amutation abolished the binding of WOW-1 Fab, a monovalentligand-mimetic anti-_v3 antibody, and theexpression of ₃ ligand-induced binding sites (LIBS)induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharpcontrast to the data obtained with _IIb, none of the mutations in the predicted W3 4-1 loop in _v impairedligand binding. These results implicate _v Y178 in ligandbinding to _v3, and they suggest thatthere are key structural differences in the adhesive ligand-bindingsites of _v3 and_IIb3.     

Fcgamma RIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia

In autoimmune hemolytic anemia (AIHA), there isaccumulating evidence for an involvement of FcR expressed byphagocytic effector cells, but demonstration of a causal relationshipbetween individual FcRs and IgG isotypes for disease development islacking. Although the relevance of IgG isotypes to human AIHA islimited, we could show a clear IgG isotype dependency in murine AIHAusing pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti-redblood cell antibodies in mice defective for FcRIII, and comparingthe clinical outcome to those in wild-type mice. FcRIII-deficientmice were completely resistent to the pathogenic effects of 105-2Hmonoclonal antibody, as shown by a lack of IgG1-mediatederythrophagocytosis in vitro and in vivo. In addition, the IgG2aresponse by 34-3C induced a less severe but persistent AIHA inFcRIII knock-out mice, as documented by a decrease in hematocrit.Blocking studies indicated that the residual anemic phenotype inducedby 34-3C in the absence of FcRIII reflects an activation of FcRIthat is normally coexpressed with FcRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcRIII and further suggest that FcRI, in addition to FcRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.     

Variable protection of beta 3-integrin--deficient mice from thrombosis initiated by different mechanisms

Platelet integrin IIb3 (GPIIb/IIIa) plays a central role inthe initiation of arterial thrombosis, but its contribution todisseminated microvascular thrombosis is less well defined. Therefore,wild-type mice (3^+/+), 3-integrin-deficient mice(3^/), and wild-type mice treated with a hamstermonoclonal antibody (1B5) that blocks murine IIb3 function weretested in models of large-vessel and microvascular thrombosis. In thelarge-vessel model, ferric chloride was used to injure the carotidartery, and the time to thrombosis was measured. In 3^+/+mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the 3^/ mice tested(P < .001). Fab and F(ab')₂ fragments of1B5 increased the median time to occlusion. To initiate systemicintravascular thrombosis, prothrombotic agents were administeredintravenously, and platelet thrombus formation was monitored by thedecrease in circulating platelet count. Three minutes after theinjection of adenosine diphosphate (ADP), collagen + epinephrine,or tissue factor, the platelet counts in 3^+/+ micedecreased by 289, 424, and 429 × 10³/µL, respectively.3^/ mice and wild-type mice pretreated with 1B5 Fab(1 mg/kg, IP) were nearly completely protected from the effects of ADP.In contrast, 3^/ mice were only partially protectedfrom the effects of collagen + epinephrine and minimally protectedfrom the effects of tissue factor. In all cases, less fibrin becamedeposited in the lungs of 3^/ mice than in wild-typemice. These results suggest that though IIb3 plays adominant role in large-vessel thrombosis, it plays a variable role insystemic intravascular thrombosis.     

Inhibition of the Differentiation of Dendritic Cells From CD34+ Progenitors by Tumor Cells: Role of Interleukin-6 and Macrophage Colony-Stimulating Factor

The escape of malignant cells from the immune response against thetumor may result from a defective differentiation or function ofprofessional antigen-presenting cells (APC), ie, dendritic cells (DC).To test this hypothesis, the effect of human renal cell carcinoma celllines (RCC) on the development of DC from CD34⁺progenitors was investigated in vitro. RCC cell lines were found torelease soluble factors that inhibit the differentiation of CD34⁺ cells into DC and trigger their commitment towardsmonocytic cells(CD14⁺CD64⁺CD1aCD86CD80HLA-DR^low)with a potent phagocytic capacity but lacking APC function. RCC CM werefound to act on the two distinct subpopulations emerging in the cultureat day 6 ([CD14⁺CD1a] and[CD14CD1a⁺]) by inhibiting thedifferentiation into DC of [CD14⁺CD1a]precursors and blocking the acquisition of APC function of the [CD14CD1a⁺] derived DC. Interleukin-6(IL-6) and macrophage colony-stimulating factor (M-CSF) were found tobe responsible for this phenomenon: antibodies against IL-6 and M-CSFabrogated the inhibitory effects of RCC CM; and recombinant IL-6and/or M-CSF inhibited the differentiation of DC similarly toRCC CM. The inhibition of DC differentiation by RCC CM was preceeded byan induction of M-CSF receptor (M-CSFR; CD115) and a loss ofgranulocyte-macrophage colony-stimulating factor receptor (GM-CSFR; CD116) expression at the surface of CD34⁺cells, two phenomenon reversed by anti-IL-6/IL-6R and anti-M-CSF antibodies, respectively. Finally, a panel of tumor cell lines producing IL-6 and M-CSF induced similar effects. Taken together, theresults suggest that the inhibition of DC development could represent afrequent mechanism by which tumor cells will escape immune recognition.     

Involvement of Na+/Ca2+ Exchanger in Inside-Out Signaling Through the Platelet Integrin alpha IIbbeta 3

The platelet integrin _IIb3 has becomea new target for the treatment of pathological thrombosis. It becomesapparent that the affinity of _IIb3 forits ligands is dynamically regulated by inside-out signaling. However,the components that couple diverse intracellular signals to thecytoplasmic domains of _IIb3 remain obscure. Employing a chymotrypsin-induced_IIb3 activation model, we previouslyproposed the hypothesis that Na⁺/Ca^{2 +}exchanger (NCX) may be involved in inside-out signaling (Shiraga et al:Blood 88:2594, 1996). In the present study, employing two unrelated Na⁺/Ca²⁺ exchange inhibitors,3,4-dichlorobenzamil (DCB) and bepridil, we investigatedthe role of NCX in platelet activation induced by various agonists indetail. Both inhibitors abolished platelet aggregation induced by allagonists examined via the inhibition of_IIb3 activation. Moreover, theseinhibitors abolished _IIb3 activationinduced by phorbol 12-myristate 13-acetate or A23187. On the otherhand, neither of these inhibitors showed apparent inhibitory effects onprotein phosphorylation of pleckstrin or myosin light chain, or anincrease in intracellular calcium ion concentrations evoked by 0.1 U/mLthrombin. These effects of the NCX inhibitors are in striking contrastto those of protein kinase C inhibitor, Ro31-8220. Biochemical andultrastructural analyses showed that NCX inhibitors, particularly DCB,made platelets "thrombasthenic". These findings suggest that theNCX is involved in the common steps of inside-out signaling throughintegrin _IIb3.     

The alpha EC Domains of Human Fibrinogen420 Contain Calcium Binding Sites But Lack Polymerization Pockets

The extended (_E) isoform unique toFibrinogen₄₂₀ (Fib₄₂₀) is distinguished fromthe conventional chain of Fibrinogen₃₄₀ by the presenceof an additional 236-residue carboxyl terminus globular domain(_EC). A recombinant form of _EC(r_EC), having a predicted mass of 27,653 Daltons, wasexpressed in yeast (Pichia pastoris) and purified by anionexchange column chromatography. Purified r_EC appears tobe predominantly intact, as judged by N-terminal sequence analysis,mass spectral analysis of the C-terminal cyanogen bromide(CNBr) fragment, and comparison of recognition byepitope-specific monoclonal antibodies. Carbohydrate determination, coupled with analysis of CNBr digestion fragments, confirms N-linked glycosylation at Asn667, the site at which sugar is attached in _E. Analysis of CNBr digestion fragments confirms thattwo disulfide bridges exist at cysteine pairs _E613/644and _E780/793. In the presence of 5 mmol/L EDTA,r_EC is highly susceptible to plasmic degradation, butCa²⁺ (5 mmol/L) renders r_EC resistant. Noprotective effect from plasmic degradation was conferred tor_EC by the peptides GPRPamide or GHRP, nor didr_EC bind to a GPR peptide column. These results suggestthat the _EC domain contains a calcium-binding site, but lacks a polymerization pocket. By analogy with the site elucidated inthe C domain, we predict that the _EC calcium bindingsite involves residues _E772-778: DADQWEE.     

Intravascular Coagulation Activation in a Murine Model of Thrombomodulin Deficiency: Effects of Lesion Size, Age, and Hypoxia on Fibrin Deposition

We consecutively inactivated both alleles of the thrombomodulin (TM)gene in murine embryonic stem (ES) cells and generated TM-deficient(TM^/) chimeric mice. Quantitation of an ES-cellmarker and protein C cofactor activity indicates that up to 50% ofpulmonary endothelial cells are ES-cell derived and therefore TMdeficient. Infusions of ¹²⁵I-fibrinogen into mice show asignificant increase (fourfold, P < .005) in radiolabeledcross-linked fibrin in TM^/ chimeric mouse lung ascompared with wild-type mice. However, only chimeric mice that exhibitat least a 30% reduction in protein C cofactor activity and are atleast 15 months old display this phenotype. Immunocytochemicallocalization of TM in chimeras shows a mosaic pattern of expression inboth large and small blood vessels. Colocalization of cross-linkedfibrin and neo (used to replace TM) reveals that fibrin is deposited inTM^/ regions. However, the fibrin deposits werelargely restricted to pulmonary vessels with a lumenal area greaterthan 100 µm². The hypercoagulable phenotype can beinduced in younger chimeric mice by exposure to hypoxia, which causes afivefold increase in -fibrin levels in lung. Our findings show thatTM chimerism results in spontaneous, intravascular fibrin depositionthat is dependent on age and the magnitude of the TM deficiency.     

Autosomal recessive chronic granulomatous disease caused by defects in NCF-1, the gene encoding the phagocyte p47-phox: mutations not arising in the NCF-1 pseudogenes

Chronic granulomatous disease (CGD) is a primary immunodeficiencycaused by defects in any one of 4 genes encoding phagocyte NADPHoxidase subunits. Unlike other CGD subtypes, in which there isgreat heterogeneity among mutations, 97% of affectedalleles in patients previously reported with A47⁰ CGDcarry a single mutation, a GT deletion (GT) in exon 2 of thep47-phox gene, NCF-1. This unusually highincidence results from recombination events between NCF-1and its highly homologous pseudogenes, in which GT originates. In 50 consecutive patients with A47⁰ CGD, 4 were identified whowere heterozygous for GT in NCF-1, and for the firsttime, 2 were identified whose DNA appeared normal at this position. Toavoid co-amplification of pseudogene sequence and to enable theidentification of mutations in these patients, allele-specificpolymerase chain reaction was used to amplify alleles not containingGT. In each of the 4 patients who were heterozygous for GT, anadditional novel mutation was identified. These were 2 missensemutations, G125  A in exon 2 (predicting Arg42  Gln)and G784  A in exon 8 (Gly262  Ser), and 2 splice junction mutations at the 5' end of intron 1, gt  at and gtg  gtt. The first of 2 patients who appeared normal at the GT position was a compound heterozygote with the G125  A transition on one allele and a deletion of G811 on the other. In the second of these patients, only a single defect was detected, G574  A,which predicts Gly192  Ser but is likely to result indefective splicing because it represents the final nucleotide of exon 6.     

Recombination Breakpoints in the Human beta -Globin Gene Cluster

The human -globin gene complex spans a region of 70 kb andcontains numerous sequence variants. These variant sites form a 5cluster (5 -haplotype) and a 3 cluster (3 -haplotype) withstrong linkage disequilibrium among the sites within each cluster, butnot between the two clusters. The 9-kb region between the 5 and 3clusters has been estimated to have rates of recombination that are 3 to 30 times normal, and the region has therefore been proposed as a`hotspot' of recombination. We describe three families with evidenceof meiotic recombination within this `hotspot' of the -globin genecluster and in which the cross-over breakpoints have been defined atthe sequence level. In one family, the recombination has occurred inthe maternal chromosome within a region of 361 bp between positions911 and 550 5 to the -globin gene. In the other two families,the recombination has occurred in the paternal chromosome within aregion of approximately 1,100 bp between positions 542 and +568relative to the -globin gene cap site. Both regions occur within the2-kb region of replication initiation (IR) in the -globin genedomain with no overlap. The IR region contains a consensus sequence fora protein (Pur), which binds preferentially tosingle-stranded DNA, a role implicated in recombination events.     

Blockage of the Early Events of Mitogenic Signaling by Interferon-gamma in Macrophages in Response to Colony-Stimulating Factor-1

Inhibition of cell proliferation is an important biologic funcphorbol ester, as measured by early gene expression, DNA tion of interferons (IFNs), which has been exploited in therasynthesis and cell proliferation. Although activation, phospeutic treatment of certain hematologic malignancies. Howphorylation, and turnover of the CSF-1 receptor and CSF-1- ever, the molecular mechanism was not clear. We have induced increase in diacylglycerol production remained norrecently shown that IFNs (/ and ) inhibit protein kinase mal, IFN- blocked CSF-1- stimulated activation of mitogen-C (PKC)-dependent (such as PDGF and phorbol ester) but activated protein kinases, Raf-1 kinase, increase in GTP-not PKC-independent (such as epidermal growth factor) actibound Ras and tyrosine phosphorylation, and activation of vation of Raf-1 and mitogen-activated protein kinases protein kinase C  (PKC-). PKC- was required for CSF-1- (MAPK/ERKs) in fibroblasts (Xu et al, Mol Cell Biol 14:8018, induced mitogenic signaling and a primary target for IFN-- 1994), suggesting a novel mechanism by which IFNs execute induced inhibition. Interestingly, although phorbol myristate their antiproliferative function. Monocytes/macrophages acetate stimulated Ras activation, PKC- did not appear to are primary targets in vivo for IFN-, the major activity of be an upstream activator of Ras. These studies clearly indimacrophage-activating factor. In the present study, mechacated that IFN- specifically inhibits PKC- activation, renism of IFN-- induced antiproliferative action in macrosulting in blockage of the early events of mitogenesis in phages in response to colony-stimulating factor- 1 (CSF-1) macrophages in response to CSF-1.     

The t(3;21) Fusion Product, AML1/Evi-1, Interacts With Smad3 and Blocks Transforming Growth Factor-beta -Mediated Growth Inhibition of Myeloid Cells

The t(3;21)(q26;q22) chromosomal translocation associated withblastic crisis of chronic myelogenous leukemia results in the formationof the AML1/Evi-1 chimeric protein, which is thought to play acausative role in leukemic transformation of hematopoietic cells. Herewe show that AML1/Evi-1 represses growth-inhibitory signaling bytransforming growth factor- (TGF-) in 32Dcl3 myeloid cells. Theactivity of AML1/Evi-1 to repress TGF- signaling depends on the twoseparate regions of the Evi-1 portion, one of which is the first zincfinger domain. AML1/Evi-1 interacts with Smad3, an intracellularmediator of TGF- signaling, through the first zinc finger domain,and represses the Smad3 activity, as Evi-1 does. We also show thatsuppression of endogenous Evi-1 in leukemic cells carrying inv(3)restores TGF- responsiveness. Taken together, AML1/Evi-1 acts as aninhibitor of TGF- signaling by interfering with Smad3 through theEvi-1 portion, and both AML1/Evi-1 and Evi-1 repress TGF--mediatedgrowth suppression in hematopoietic cells. Thus, AML1/Evi-1 maycontribute to leukemogenesis by specifically blocking growth-inhibitorysignaling of TGF- in the t(3;21) leukemia.     

CBFbeta -SMMHC, Expressed in M4eo Acute Myeloid Leukemia, Reduces p53 Induction and Slows Apoptosis in Hematopoietic Cells Exposed to DNA-Damaging Agents

CBF-SMMHC is expressed in M4Eo acute myeloid leukemia (AML) as aresult of inv(16), but how it contributes to leukemogenesis isunknown. p53 mutations are rare in de novo AML, but theyare common in many malignancies. Expression of CBF-SMMHC in Ba/F3 cells reduced p53 induction in response to ionizing radiation oretoposide 3- to 4-fold. However, p53 induction was normal in Ba/F3cells expressing a CBF-SMMHC variant that does not interfere withDNA binding by CBF, indicating that a CBF genetic target regulates p53induction. The p53 gene may be regulated by CBF, because p53 mRNAlevels were reduced by CBF-SMMHC. Reduced p53 induction was notcaused by slowed cell proliferation, a consequence of CBF-SMMHCexpression, because p53 was induced similarly in control cultures andin cultures propagated in 10-fold less interleukin-3 (IL-3).CBF-SMMHC did not slow apoptosis resulting from IL-3 withdrawal,where p53 induction is minimal, but slowed apoptosis in Ba/F3 cellsexposed to 10 Gy of ionizing radiation or 3 to 8 µg/mL etoposide,providing 2-fold protection at 6 or 18 hours. Inhibition of apoptosiswas temporary, because all the cells exposed to these doses ultimatelydied, and clonal survival assays performed using 0.04 µg/mL etoposidedid not show protection by CBF-SMMHC. p21 levels were increased incells subjected to DNA damage, regardless of CBF-SMMHC expressionand attenuated p53 induction. Bcl-2, bcl-x_L,bcl-x_S, and bax levels were unaffected by CBF-SMMHC. Attenuated p53 induction may contribute to leukemogenesis byCBF-SMMHC by slowing apoptosis via a p21-independent mechanism.     

Ristocetin-dependent, but not botrocetin-dependent, binding of von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with shear-dependent interactions

Under conditions of high shear stress, both hemostasis andthrombosis are initiated by the interaction of the platelet membrane glycoprotein (GP) Ib-IX-V complex with its adhesive ligand, von Willebrand factor (vWF), in the subendothelial matrix or plasma. Thisinteraction involves the A1 domain of vWF and the N-terminal extracellular region of GP Ib (His-1-Glu-282), and it can also beinduced under static conditions by the modulators ristocetin andbotrocetin. In this study, a panel of anti-vWF and anti-GP Ibantibodiespreviously characterized for their effects on ristocetin- and botrocetin-dependent vWF-GP Ib-IX-V interactionswas analyzed fortheir capacity to inhibit either the adhesion of Chinese hamster ovarycells expressing recombinant GP Ib to surface-associated vWF underhydrodynamic flow or shear-stress-induced platelet aggregation. Thecombined results suggest that the shear-dependent interactions betweenvWF and GP Ib closely correlate with ristocetin- rather thanbotrocetin-dependent binding under static conditions and that certainanti-vWF monoclonal antibodies are able to selectively inhibitshear-dependent platelet aggregation.