An essential regulatory role for macrophage migration inhibitory factor in T-cell activation.

The protein known as macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered and was described 30 years ago to be a T-cell-derived factor that inhibited the random migration of macrophages in vitro. A much broader role for MIF has emerged recently as a result of studies that have demonstrated it to be released from the anterior pituitary gland in vivo. MIF also is the first protein that has been identified to be secreted from monocytes/macrophages upon glucocorticoid stimulation. Once released, MIF acts to "override" or counter-regulate the suppressive effects of glucocorticoids on macrophage cytokine production. We report herein that MIF plays an important regulatory role in the activation of T cells induced by mitogenic or antigenic stimuli. Activated T cells produce MIF and neutralizing anti-MIF antibodies inhibit T-cell proliferation and interleukin 2 production in vitro, and suppress antigen-driven T-cell activation and antibody production in vivo. T cells also release MIF in response to glucocorticoid stimulation and MIF acts to override glucocorticoid inhibition of T-cell proliferation and interleukin 2 and interferon gamma production. These studies indicate that MIF acts in concert with glucocorticoids to control T-cell activation and assign a previously unsuspected but critical role for MIF in antigen-specific immune responses.

Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis.

Live vaccine vectors are usually very effective and generally elicit immune responses of higher magnitude and longer duration than nonliving vectors. Consequently, much attention has been turned to the engineering of oral pathogens for the delivery of foreign antigens to the gut-associated lymphoid tissues. However, no bacterial vector has yet been designed to specifically take advantage of the nasal route of mucosal vaccination. Herein we describe a genetic system for the expression of heterologous antigens fused to the filamentous hemagglutinin (FHA) in Bordetella pertussis. The Schistosoma mansoni glutathione S-transferase (Sm28GST) fused to FHA was detected at the cell surface and in the culture supernatants of recombinant B. pertussis. The mouse colonization capacity and autoagglutination of the recombinant microorganism were indistinguishable from those of the wild-type strain. In addition, and in contrast to the wild-type strain, a single intranasal administration of the recombinant strain induced both IgA and IgG antibodies against Sm28GST and against FHA in the bronchoalveolar lavage fluids. No anti-Sm28GST antibodies were detected in the serum, strongly suggesting that the observed immune response was of mucosal origin. This demonstrates, to our knowledge, for the first time that recombinant respiratory pathogens can induce mucosal immune responses against heterologous antigens, and this may constitute a first step toward the development of combined live vaccines administrable via the respiratory route.

A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.

Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element.

Notch is a transmembrane receptor that plays a critical role in cell fate determination. In Drosophila, Notch binds to and signals through Suppressor of Hairless. A mammalian homologue of Suppressor of Hairless, named CBF1 (or RBPJk), is a ubiquitous transcription factor whose function in mammalian Notch signaling is unknown. To determine whether mammalian Notch can stimulate transcription through a CBF1-responsive element (RE), we cotransfected a CBF1-RE-containing chloramphenicol acetyltransferase reporter and N1(deltaEC), a constitutively active form of human Notch1 lacking the extracellular domain, into DG75, COS-1, HeLa, and 293T cells, which all contain endogenous CBF1. N1(deltaEC) dramatically increased chloramphenicol acetyltransferase activity in these cells, indicating functional coupling of Notch1 and CBF1. The activity was comparable to that produced by the Epstein-Barr virus protein EBNA2, a well-characterized, potent transactivator of CBF1. To test whether CBF1 and Notch1 interact physically, we tagged CBF1 with an epitope from the influenza virus hemagglutinin or with the N-terminal domain of gal4, and transfected the tagged CBF1 plus N1(deltaEC) into COS-1 cells. Cell lysates were immunoprecipitated and immunoblotted with several anti-Notch1 antibodies [to detect N1(deltaEC)] or with antibodies to hemagglutinin or gal4 (to detect CBF1). Each immunoprecipitate contained a complex of N1(deltaEC) and CBF1. In summary, we find that the truncated, active form of human Notch1, N1(deltaEC), binds CBF1 and activates transcription through a CBF1-RE-containing promoter. We conclude that CBF1 is a critical downstream protein in the human Notch1 signaling pathway.

Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.

Interleukin (IL) 15/IL-T production by the adult T-cell leukemia cell line HuT-102 is associated with a human T-cell lymphotrophic virus type I region /IL-15 fusion message that lacks many upstream AUGs that normally attenuates IL-15 mRNA translation.

We reported previously that the human T-cell lymphotrophic virus type I (HTLV-I)-associated adult T-cell leukemia line HuT-102 produces a cytokine designated interleukin (IL) T that requires interleukin (IL) 2 receptor beta-subunit expression for its action. Using anti-cytokine antibodies, we demonstrated that IL-T is identical to the simultaneously described IL-15. When compared to activated monocytes, IL-15 mRNA expression was 6- to 10-fold greater in HuT-102 cells. The predominant IL-15 message from HuT-102 is a chimeric mRNA joining a segment of the R region of the long terminal repeat of HTLV-I and the 5'-untranslated region (UTR) of IL-15. Normally, by alternative splicing, this 118-nucleotide R element represents the most 5' region of several HTLV-I transcripts including tax, rex, and env. The introduction of the R element eliminated over 200 nucleotides of the IL-15 5'-UTR, including 8 of 10 upstream AUGs that are present in normal IL-15 messages. On analysis of the 5'-UTR of normal IL-15, we demonstrated that the presence of these 10 upstream AUGs interferes with IL-15 mRNA translation. Thus, IL-15 synthesis by the adult T-cell leukemia line HuT- 102 involves an increase in IL-15 mRNA transcription and translation secondary to the production of an HTLV-I R element fusion message that lacks many upstream AUGs.

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

Enhanced and accelerated lymphoproliferation in Fas-null mice.

Fas is a 45-kDa membrane protein that transduces an apoptotic signal. The mouse lymphoproliferation (lpr) mutation is a leaky mutation of Fas. In this study, we examined lymphocyte development in Fas-null mice generated by gene targeting. The Fas-/- mice progressively accumulated abnormal T cells (Thy1+, B220+, CD4-, and CD8-) and developed lymphadenopathy and splenomegaly, which were much more accelerated and pronounced than those in lpr mice. In addition, the Fas-null mice showed lymphocytosis, accompanied by lymphocytic infiltration in the lungs and liver. The number of apparently normal B cells also increased, and large amounts of immunoglobulins, including anti-DNA antibodies, were produced. Thymic clonal deletion, assessed by deletion of T cells reactive to mouse endogenous superantigens, was apparently normal in the Fas-/- mice, whereas the peripheral clonal deletion of mature T cells against a bacterial superantigen was impaired. These results suggested that Fas plays a decisive role in peripheral clonal deletion but not in negative selection in the thymus.

Poliovirus chimeras replicating under the translational control of genetic elements of hepatitis C virus reveal unusual properties of the internal ribosomal entry site of hepatitis C virus.

Chimeric genomes of poliovirus (PV) have been constructed in which the cognate internal ribosomal entry site (IRES) element was replaced by genetic elements of hepatitis C virus (HCV). Replacement of PV IRES with nt 9-332 of the genotype Ib HCV genome, a sequence comprising all but the first eight residues of the 5' nontranslated region (5'NTR) of HCV, resulted in a lethal phenotype. Addition of 366 nt of the HCV core-encoding sequence downstream of the HCV 5'NTR yielded a viable PV/HCV chimera, which expressed a stable, small-plaque phenotype. This chimeric genome encoded a truncated HCV core protein that was fused to the N terminus of the PV polyprotein via an engineered cleavage site for PV proteinase 3CPpro. Manipulation of the HCV core-encoding sequence of this viable chimera by deletion and frameshift yielded results suggesting that the 5'-proximal sequences of the HCV open reading frame were essential for viability of the chimera and that the N-terminal basic region of the HCV core protein is required for efficient replication of the chimeric virus. These data suggest that the bona fide HCV IRES includes genetic information mapping to the 5'NTR and sequences of the HCV open reading frame. PV chimeras replicating under translational control of genetic elements of HCV can serve to study HCV IRES function in vivo and to search for anti-HCV chemotherapeutic agents.

Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus erythematosus.

The restriction of phosphatidylserine (PtdSer) to the inner surface of the plasma membrane bilayer is lost early during apoptosis. Since PtdSer is a potent surface procoagulant, and since there is an increased incidence of coagulation events in patients with systemic lupus erythematosus (SLE) who have anti-phospholipid antibodies, we addressed whether apoptotic cells are procoagulant and whether anti-phospholipid antibodies influence this. Apoptotic HeLa cells, human endothelial cells, and a murine pre-B-cell line were markedly procoagulant in a modified Russell viper venom assay. This procoagulant effect was entirely abolished by addition of the PtdSer-binding protein, annexin V, confirming that it was PtdSer-dependent. The procoagulant effect was also abolished by addition of IgG purified from the plasma of three patients with anti-phospholipid antibody syndrome, but not IgG from normal controls. Confocal microscopy of apoptotic cells stained with fluorescein-isothiocyanate-conjugated-annexin V demonstrated (Ca2+)-dependent binding to the surface of membrane blebs o apoptotic cells, but not to intracellular membranes. Recent data indicate that the surface blebs of apoptotic cells constitute an important immunogenic particle in SLE. We propose that the PtdSer exposed on the outside of these blebs can induce the production of anti-phospholipid antibodies, which might also enhance the immunogenicity of the bleb contents. When apoptosis occurs in a microenvironment in direct contact with circulating plasma, the unique procoagulant consequences of the apoptotic surface may additionally be expressed. This might explain the increased incidence of pathological intravascular coagulation events that occur in some lupus patients who have anti-phospholipid antibodies.

Isolation and characterization of a cell surface albumin-binding protein from vascular endothelial cells.

Albumin-binding proteins identified in vascular endothelial cells have been postulated to contribute to the transport of albumin via a process involving transcytosis. In the present study, we have purified and characterized a 57- to 60-kDa (gp60) putative albumin-binding protein from bovine pulmonary microvessel endothelial cells. The endothelial cell membranes were isolated from cultured cells by differential centrifugation and solubilized with sodium cholate and urea. The solubilized extract was concentrated after dialysis by ethanol precipitation and reextracted with Triton X-100, and the resulting extract was subjected to DEAE-cellulose column chromatography. Proteins eluted from this column were further separated using preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis and used for immunizing rabbits. Fluorescence-activated cell sorter analysis using the anti-gp60 antibodies demonstrated the expression of gp60 on the endothelial cell surface. Affinity-purified anti-gp60 antibodies inhibited approximately 90% of the specific binding of 125I-labeled albumin to bovine pulmonary microvessel endothelial cell surface. The anti-gp60 antibodies reacted with gp60 from bovine pulmonary artery, bovine pulmonary microvessel, human umbilical vein, and rat lung endothelial cell membranes. Bovine anti-gp60 antibodies also reacted with bovine secreted protein, acidic and rich in cysteine (SPARC). However, bovine SPARC NH2-terminal sequence (1-56 residues) antibodies did not react with gp60, indicating that the endothelial cell-surface-associated albumin-binding protein gp60 was different from the secreted albumin-binding protein SPARC. We conclude that the endothelial cell-surface-associated gp60 mediates the specific binding of native albumin to endothelial cells and thus may regulate the uptake of albumin and its transcytosis.

Cloning and expression of a gene encoding an integrin-like protein in Candida albicans.

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.

The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion.

Although the CD34 antigen is widely used in the identification and purification of hemopoietic stem and progenitor cells, its function within hemopoiesis is unknown. We have investigated this issue by ectopically expressing human (hu) CD34 on the surface of murine hemopoietic cells. Forced expression of hu-CD34 in the thymocytes of transgenic mice did not appear to affect the development, maturation, or distribution of murine T cells but did significantly increase their ability to adhere to bone marrow stromal layers of human but not mouse origin. Ectopic expression of hu-CD34 on murine 416B cells, a multipotential progenitor that expresses murine CD34, yielded similar results. In both cases hu-CD34-dependent adhesion was enhanced by molecular engagement of the hu-CD34 protein using anti-CD34 antibodies. These results provide evidence that CD34 promotes the adhesive interactions of hemopoietic cells with the stromal microenvironment of the bone marrow thereby implicating CD34 in regulation and compartmentalization of stem cells. We propose that CD34 regulates these processes in part via an indirect mechanism, signaling changes in cellular adhesion in response to molecular recognition of an as yet unidentified stromal CD34 counterreceptor or ligand.

Major histocompatibility complex class II DR alleles DRB1*1501 and those encoding HLA-DR13 are preferentially associated with a diminution in maternally transmitted human immunodeficiency virus 1 infection in different ethnic groups: determination by an automated sequence-based typing method.

Transmission of human immunodeficiency virus 1 (HIV-1) from an infected women to her offspring during gestation and delivery was found to be influenced by the infant's major histocompatibility complex class II DRB1 alleles. Forty-six HIV-infected infants and 63 seroreverting infants, born with passively acquired anti-HIV antibodies but not becoming detectably infected, were typed by an automated nucleotide-sequence-based technique that uses low-resolution PCR to select either the simpler Taq or the more demanding T7 sequencing chemistry. One or more DR13 alleles, including DRB1*1301, 1302, and 1303, were found in 31.7% of seroreverting infants and 15.2% of those becoming HIV-infected [OR (odds ratio) = 2.6 (95% confidence interval 1.0-6.8); P = 0.048]. This association was influenced by ethnicity, being seen more strongly among the 80 Black and Hispanic children [OR = 4.3 (1.2-16.4); P = 0.023], with the most pronounced effect among Black infants where 7 of 24 seroreverters inherited these alleles with none among 12 HIV-infected infants (Haldane OR = 12.3; P = 0.037). The previously recognized association of DR13 alleles with some situations of long-term nonprogression of HIV suggests that similar mechanisms may regulate both the occurrence of infection and disease progression after infection. Upon examining for residual associations, only only the DR2 allele DRB1*1501 was associated with seroreversion in Caucasoid infants (OR = 24; P = 0.004). Among Caucasoids the DRB1*03011 allele was positively associated with the occurrence of HIV infection (P = 0.03).

In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA-binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus.