An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes

CD22 is a B cell-restricted glycoprotein involved in signal transduction and modulation of cellular activation. It is also an I-type lectin (now designated Siglec-2), whose extracellular domain can specifically recognize α2–6-linked sialic acid (Sia) residues. This activity is postulated to mediate intercellular adhesion and/or to act as a coreceptor in antigen-induced B cell activation. However, studies with recombinant CD22 indicate that the lectin function can be inactivated by expression of α2–6-linked Sia residues on the same cell surface. To explore whether this masking phenomenon affects native CD22 on B cells, we first developed a probe to detect the lectin activity of recombinant CD22 expressed on Chinese hamster ovary cells (which have no endogenous α2–6-linked Sia residues). This probe is inactive against CD22-positive B lymphoma cells and Epstein–Barr virus-transformed lymphoblasts which express high levels of α2–6-linked Sia residues. Enzymatic desialylation unmasks the CD22 lectin activity, indicating that endogenous Sia residues block the CD22 lectin-binding site. Truncation of the side chains of cell surface Sia residues by mild periodate oxidation (known to abrogate Sia recognition by CD22) also had this unmasking effect, indicating that the effects of desialylation are not due to a loss of negative charge. Normal resting B cells from human peripheral blood gave similar findings. However, the lectin is partially unmasked during in vitro activation of these cells. Thus, the lectin activity of CD22 is restricted by endogenous sialylation in resting B cells and may be transiently unmasked during in vivo activation, perhaps to modulate intercellular or intracellular interactions at this critical stage in the humoral response.

Transfer of human serum IgG to nonobese diabetic Igμnull mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren’s syndrome

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sjögren’s syndrome. NOD.Igμnull mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igμnull mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab′)2 fragments from parental NOD mice or human primary Sjögren’s syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sjögren’s syndrome.

Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin⋅FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.

Cell-cycle arrest and apoptosis hypersusceptibility as a consequence of Lck deficiency in nontransformed T lymphocytes

By using antisense RNA, Lck-deficient transfectants of a T helper 2 (Th2) clone have been derived and shown to have a qualitative defect in the T cell receptor signaling pathway. A striking feature observed only in Lck-deficient T cells was the presence of a constitutively tyrosine-phosphorylated 32-kDa protein. In the present study, we provide evidence that this aberrantly hyperphosphorylated protein is p34cdc2 (cdc2) a key regulator of cell-cycle progression. Lck-deficient transfectants expressed high levels of cdc2 protein and its regulatory units, cyclins A and B. The majority of cdc2, however, was tyrosine-phosphorylated and therefore enzymatically inactive. The transfectants were significantly larger than the parental cells and contained 4N DNA. These results establish that a deficiency in Lck leads to a cell-cycle arrest in G2. Moreover, transfected cells were hypersusceptible to apoptosis when activated through the T cell receptor. Importantly, however, this hypersusceptibility was largely reversed in the presence of T cell growth factors. These findings provide evidence that, in mature T lymphocytes, cell-cycle progression through the G2–M check point requires expression of the Src-family protein tyrosine kinase, Lck. This requirement is Lck-specific; it is observed under conditions in which the closely related Fyn kinase is expressed normally, evincing against a redundancy of function between these two kinases.

Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to the Idd5 diabetes susceptibility region

Defects in lymphocyte apoptosis may lead to autoimmune disorders and contribute to the pathogenesis of type 1 diabetes. Lymphocytes of nonobese diabetic (NOD) mice, an animal model of autoimmune diabetes, have been found resistant to various apoptosis signals, including the alkylating drug cyclophosphamide. Using an F2 intercross between the apoptosis-resistant NOD mouse and the apoptosis-susceptible C57BL/6 mouse, we define a major locus controlling the apoptosis-resistance phenotype and demonstrate its linkage (logarithm of odds score = 3.9) to a group of medial markers on chromosome 1. The newly defined gene cannot be dissociated from Ctla4 and Cd28 and in fact marks a 20-centimorgan region encompassing Idd5, a previously postulated diabetes susceptibility locus. Interestingly, we find that the CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and the CD28 costimulatory molecules are defectively expressed in NOD mice, suggesting that one or both of these molecules may be involved in the control of apoptosis resistance and, in turn, in diabetes susceptibility.

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

Acknowledgements The work was in part funded by UK Medical Research Council project grant G0601253 to G.S.B. and R.W.B.

Expression of HLA class I-specific inhibitory natural killer cell receptors in HIV-specific cytolytic T lymphocytes: Impairment of specific cytolytic functions

Human T lymphocytes have been shown to express inhibitory natural killer cell receptors (NKR), which can down-regulate T cell antigen receptor-mediated T cell function, including cytolytic activity. In the present study, we demonstrate that CD3+NKR+ cells can be identified in HIV-infected patients. HIV-specific cytolytic activity was analyzed in five patients in whom autologous lymphoblastoid B cell lines could be derived as a source of autologous target cells. Phytohemagglutinin-activated T cell populations that had been cultured in interleukin 2 displayed HIV-specific cytotoxic T lymphocyte (CTL) activity against HIV env, gag, pol, and nef in 3 of 5 patients. Addition of anti-NKR mAb of IgM isotype could increase the specific CTL activity. Moreover, in one additional patient, HIV-specific CTL activity was undetectable; however, after addition of anti-NKR mAb such CTL activity appeared de novo. Similar results were obtained by analysis of CD3+NKR+ clones derived from two patients. These data provide direct evidence that CD3+NKR+ cells may include antigen (HIV)-specific CTLs and that mAb-mediated masking of inhibitory NKR may revert the down-regulation of CTL function.

Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-HbHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4+ cells, and this protective effect was observed only in the PBL derived from donors having a CD4+ cell count above 200 per mm3. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.

Growth and viability of macrophages continuously stimulated to produce nitric oxide

Deregulated production of nitric oxide (NO) has been implicated in the development of certain human diseases, including cancer. We sought to assess the damaging potential of NO produced under long-term conditions through the development of a suitable model cell culture system. In this study, we report that when murine macrophage-like RAW264.7 cells were exposed continuously to bacterial lipopolysaccharide (LPS) or mouse recombinant interferon-γ (IFN-γ) over periods of 21–23 days, they continued to grow, but with doubling times 2 to 4 times, respectively, longer than the doubling time of unstimulated cells. Stimulated cells produced NO at rates of 30 to 70 nmol per million cells per day throughout the stimulation period. Within 24 hr after removal of stimulant, cells resumed exponential growth. Simultaneous exposure to LPS and IFN-γ resulted in decreased cell number, which persisted for 2 days after removal of the stimulants. Exponential growth was attained only after an additional 4 days. Addition of N-methyl-l-arginine (NMA), an NO synthase inhibitor, to the medium inhibited NO production by 90% of all stimulated cells, partially reduced doubling time of cells stimulated with LPS or IFN-γ, and partially increased viability and growth rates in those exposed to both LPS and IFN-γ. However, when incubated with LPS and IFN-γ at low densities both in the presence and in the absence of NMA, cells grew at a rate slower than that of unstimulated cells, with no cell death, and they resumed exponential growth 24 hr after removal of stimulants. Results from cell density experiments suggest that macrophages are protected from intracellularly generated NO; much of the NO damaging activity occurred outside of the producer cells. Collectively, results presented in this study suggest that the type of cellular toxicity observed in macrophages is markedly influenced by rate of exposure to NO: at low rates of exposure, cells exhibit slower growth; at higher rates, cells begin to die; at even higher rates, cells undergo growth arrest or die. The ability of RAW264.7 cells to produce NO over many cell generations makes the cell line a useful system for the study of other aspects of cellular damage, including genotoxicity, resulting from exposure to NO under long-term conditions.

Adenoviral-mediated gene transfer in lymphocytes

Although adenovirus can infect a wide range of cell types, lymphocytes are not generally susceptible to adenovirus infection, in part because of the absence of the expression of the cellular receptor for the adenoviral fiber protein. The cellular receptor for adenovirus and coxsackievirus (CAR) recently was cloned and shown to mediate adenoviral entry by interaction with the viral fiber protein. We show that the ectopic expression of CAR in various lymphocyte cell lines, which are almost completely resistant to adenovirus infection, is sufficient to facilitate the efficient transduction of these cells by recombinant adenoviruses. Furthermore, this property of CAR does not require its cytoplasmic domain, consistent with the idea that CAR primarily serves as a high affinity binding site for the adenoviral fiber protein, and that viral entry is mediated by interaction of the viral penton base proteins with cellular integrins. As a demonstration of their functional utility, we used CAR-expressing lymphocytes transduced with an adenovirus expressing Fas ligand to efficiently kill Fas receptor-expressing tumor cells. The ability to efficiently manipulate gene expression in lymphocyte cells by using adenovirus vectors should facilitate the functional characterization of pathways affecting lymphocyte physiology.

Cloning of a gene (RIG-G) associated with retinoic acid-induced differentiation of acute promyelocytic leukemia cells and representing a new member of a family of interferon-stimulated genes

In a cell line (NB4) derived from a patient with acute promyelocytic leukemia, all-trans-retinoic acid (ATRA) and interferon (IFN) induce the expression of a novel gene we call RIG-G (for retinoic acid-induced gene G). This gene codes for a 58-kDa protein containing 490 amino acids with several potential sites for post-translational modification. In untreated NB4 cells, the expression of RIG-G is undetectable. ATRA treatment induces the transcriptional expression of RIG-G relatively late (12–24 hr) in a protein synthesis-dependent manner, whereas IFN-α induces its expression early (30 min to 3 hr). Database search has revealed a high-level homology between RIG-G and several IFN-stimulated genes in human (ISG54K, ISG56K, and IFN-inducible and retinoic acid-inducible 58K gene) and some other species, defining a well conserved gene family. The gene is composed of two exons and has been mapped by fluorescence in situ hybridization to chromosome 10q24, where two other human IFN-stimulated gene members are localized. A synergistic induction of RIG-G expression in NB4 cells by combined treatment with ATRA and IFNs suggests that a collaboration exists between their respective signaling pathways.

Peptide-specific cytotoxic T lymphocytes restricted by nonself major histocompatibility complex class I molecules: Reagents for tumor immunotherapy

Studies in melanoma patients have revealed that self proteins can function as targets for tumor-reactive cytotoxic T lymphocytes (CTL). One group of self proteins MAGE, BAGE, and GAGE are normally only expressed in testis and placenta, whilst another group of CTL recognized proteins are melanocyte-specific differentiation antigens. In this study we have investigated whether CTL can be raised against a ubiquitously expressed self protein, mdm-2, which is frequently overexpressed in tumors. The observation that T-cell tolerance is self major histocompatibility complex-restricted was exploited to generate CTL specific for an mdm-2 derived peptide presented by nonself major histocompatibility complex class I molecules. Thus, the allo-restricted T-cell repertoire of H-2d mice was used to isolate CTL specific for the mdm100 peptide presented by allogeneic H-2Kb class I molecules. In vitro, these CTL discriminated between transformed and normal cells, killing specifically Kb-positive melanoma and lymphoma tumors but not Kb-expressing dendritic cells. In vivo, the CTL showed antitumor activity and delayed the growth of melanoma as well as lymphoma tumors in H-2b recipient mice. These experiments show that it is possible to circumvent T-cell tolerance to ubiquitously expressed self antigens, and to target CTL responses against tumors expressing elevated levels of structurally unaltered proteins.

Interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppression in vitro

A marked suppression of immune function has long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles. As a hallmark of measles virus (MV)-induced immunosuppression, peripheral blood lymphocytes (PBLs) isolated from patients exhibit a significantly reduced capacity to proliferate in response to mitogens, allogens, or recall antigens. In an in vitro system we show that proliferation of naive PBLs [responder cells (RCs)] in response to a variety of stimuli was significantly impaired after cocultivation with MV-infected, UV-irradiated autologous PBLs [presenter cells (PCs)]. We further observed that a 50% reduction in proliferation of RCs could still be observed when the ratio of PC to RC was 1:100. The effect was completely abolished after physical separation of the two populations, which suggests that soluble factors were not involved. Proliferative inhibition of the RCs was observed after short cocultivation with MV-infected cells, which indicates that surface contact between one or more viral proteins and the RC population was required. We identified that the complex of both MV glycoproteins, F and H, is critically involved in triggering MV-induced suppression of mitogen-dependent proliferation, since the effect was not observed (i) using a recombinant MV in which F and H were replaced with vesicular stomatitis virus G or (ii) when either of these proteins was expressed alone. Coexpression of F and H, however, lead to a significant proliferative inhibition in the RC population. Our data indicate that a small number of MV-infected PBLs can induce a general nonresponsiveness in uninfected PBLs by surface contact, which may, in turn, account for the general suppression of immune responses observed in patients with acute measles.

Altered peptide ligands act as partial agonists by inhibiting phospholipase C activity induced by myasthenogenic T cell epitopes

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195–212 and p259–271, previously were shown to stimulate the proliferation of peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid-substituted analogs of p195–212 and p259–271, as well as a dual analog composed of the tandemly arranged two single analogs, were shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Stimulation of T cells through the antigen-specific T cell receptor activates tyrosine kinases and phospholipase C (PLC). Therefore, in attempts to understand the mechanism of action of the analogs, we first examined whether the myasthenogenic peptides trigger tyrosine phosphorylation and activation of phospholipase C. For that purpose, we measured generation of inositol phosphates and tyrosine phosphorylation of PLC after stimulation of the p195–212- and p259–271-specific T cell lines with these myasthenogenic peptides. Both myasthenogenic peptides stimulated generation of inositol phosphates as well as tyrosine phosphorylation of PLC. However, the single and dual analogs, although inducing tyrosine phosphorylation of PLC, could not induce PLC activity. Furthermore, the single and dual analogs inhibited the induced PLC activity whereas they could not inhibit tyrosine phosphorylation of PLC that was caused by the myasthenogenic peptides. Thus, the altered peptides and the dual analog act as partial agonists. The down-regulation of PLC activity by the analogs may account for their capacity to inhibit in vitro MG-associated T cell responses.