Phylogenetic analysis of platelet-derived growth factor by radio-receptor assay.

Competition between 125I-labeled platelet-derived growth factor (PDGF) and unlabeled PDGF forms the basis of a specific "radio-receptor assay" for quantifying PDGF in clotted blood serum. Human clotted blood serum contains 15 ng/ml of PDGF by radio-receptor assay; this corresponds to a PDGF content of approximately 7.5 x 10(-5) pg per circulating platelet, a figure which is corroborated by purification data. Clotted blood sera from mammals, lower vertebrates and marine invertebrates were screened for homologues of human PDGF by radio-receptor assay. All tested specimens from phylum Chordata contain a mitogenic agent that competes with human PDGF for receptor binding. Sera from tunicates down on the chordate line of evolution and sera from all tested animals on the arthropod line of development were negative. The phylogenetic distribution of PDGF homologue does not correlate with platelet distribution since platelets and their precursor cell--the bone marrow megacaryocyte--are unique to the mammalian hematopoietic system. One anatomical feature appearing coordinately with PDGF on the vertebrate line of development is a pressurized circulatory system. The coincidental appearance of these features may lend support to the hypothesis that PDGF plays a role in maintenance and repair of the vascular lining in vivo.

Persistence of the mitogenic response to platelet-derived growth factor (competence) does not reflect a long-term interaction between the growth factor and the target cell.

Quiescent BALB/c-3T3 cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA even if PDGF is removed from cell culture medium prior to the onset of DNA synthesis. We have suggested that persistence of the PDGF-induced competent state reflects a rapidly induced and relatively stable biochemical change within the target cells. Others suggest that the phenomenon reflects a long-term association between PDGF and its target cells or perhaps between PDGF and the cell culture dish. This controversy has been addressed (a) by examining the effect of anti-PDGF antibodies on PDGF-induced competence and (b) by studying the chemical fate of 125I-labeled PDGF. Anti-PDGF antibodies inactive both soluble and surface-bound PDGF. However, if quiescent 3T3 cells are exposed to PDGF for as little as 30 min, subsequent addition of these antibodies to the culture medium does not prevent the mitogenic response. Under conditions where the PDGF-induced competent state decays stochastically with a t1/2 of 18-20 h, cell-associated 125I-PDGF decays with a t1/2 of approximately 50 min. These data do not support the concept that persistence of the PDGF-induced competent state reflects a long-term association between PDGF and the target cells or between PDGF and the culture dish.

Variants of the human high-affinity receptor (Fc gamma RI) for immunoglobulin G.

PCR-amplification cloning of the cDNA encoding the human high-affinity receptor for IgG (Fc gamma RI) revealed two splice variants which coincide with domain boundaries predicted by amino acid sequence comparison. Both splice variants maintain the open reading frame.

Cytokines and HIV envelope glycoprotein gp120 stimulate Na+/H+ exchange in astrocytes.

The pathogenesis of the human immunodeficiency virus (HIV)-associated cognitive/motor complex, or acquired immunodeficiency syndrome (AIDS) dementia complex, is unknown, but it afflicts over 50% of all patients infected with HIV-1. Because neurons are not directly infected with HIV-1, the causes of neuronal dysfunction are undoubtedly indirect. We investigated the role of the astrocyte in the development of AIDS dementia complex, focusing on cytokine and HIV-1 gp120 stimulation of Na+/H+ exchange (NHE) activity of primary rat astrocytes. Our results show that the cytokines tumor necrosis factor-alpha, interferon (IFN)-gamma, and interleukin (IL)-1 beta (all found to be elevated in the central nervous system of AIDS patients), can stimulate Na+/H+ exchange, but that transforming growth factor-beta, IL-2, and IL-6 do not. IFN-gamma and gp120-induced activation of Na+/H+ exchange appears to be mediated through activation of tyrosine kinase (TK), because TK inhibitors block the action of IFN-gamma and gp120. Additionally, gp120 induces tyrosine phosphorylation of two proteins (approximately 90 and 130 kDa), which is also inhibited by TK inhibitors. The predominant NHE isoform present in rat astrocytes is NHE-1; however, other isoforms are also present. We conclude that Na+/H+ exchange of rat astrocytes can be differentially stimulated by cytokines and HIV-1 gp120. We hypothesize that the resultant increase in intracellular pH with its concomitant changes in astrocyte membrane permeability properties produces an imbalance in the K+ and glutamate microenvironment of the neurons, leading to a rise in intraneuronal Ca2+ and eventual neuronal dysfunction and/or demise.

Human immunodeficiency virus type 2 envelope glycoprotein: differential CD4 interactions of soluble gp120 versus the assembled envelope complex.

Utilizing a recombinant vaccinia expression system, we investigated the biological properties and CD4 receptor interactions of the envelope glycoproteins of a noncytopathic human immunodeficiency virus type 2 strain, termed HIV-2/ST, and a highly cytopathic variant derived from it. The efficiency and host cell range of syncytium formation by the recombinant glycoproteins of both viruses were highly restricted compared to those of prototypic strains of HIV (HIV-2/ROD or HIV-1/IIIB). However, the glycoprotein of cytopathic but not wild-type ST generated numerous large syncytia in the human T-cell line Sup T1 from which it was derived. A single cell line (Molt 4 clone 8) was permissive to fusion by both wild-type and cytopathic ST envelopes, but only the glycoprotein of cytopathic ST could be inhibited with a soluble form of the viral receptor CD4 (sCD4). While these results indicated major differences in the envelope glycoprotein-CD4 receptor interactions of wild-type versus cytopathic ST, direct and competition binding assays utilizing soluble external glycoprotein (SU) and sCD4 surprisingly revealed equivalent low binding affinity for both viruses. From these experiments we conclude that relevant biological properties (e.g., CD4 binding, cytopathic potential, and sCD4 neutralization) of HIV viruses which differ in their pathogenic potential are reflected in the sCD4 interactions of the assembled native envelope complex (as on cell or virion surfaces) but not the soluble SU glycoprotein.

Envelope glycoprotein gp120 of human immunodeficiency virus type 1 alters ion transport in astrocytes: implications for AIDS dementia complex.

Infection by human immunodeficiency virus type 1 (HIV-1) is often complicated by a variety of neurological abnormalities. The most common clinical syndrome, termed acquired immunodeficiency syndrome (AIDS) dementia complex, presents as a subcortical dementia with cognitive, motor, and behavioral disturbances and is unique to HIV-1 infection. The pathogenesis of this syndrome is poorly understood but is believed to involve interactions among virally infected macrophages/microglia, astrocytes, and neurons. In this study, we show that exposure of primary rat and human astrocytes to heat-activated HIV-1 virions, or to eukaryotically expressed HIV-1 and HIV-2 envelope glycoproteins (gp120) stimulates amiloride-sensitive Na+/H+ antiport, potassium conductance, and glutamate efflux. These effects are blocked specifically by amiloride, an inhibitor of Na+/H+ antiport and by the selective removal of gp120 with immobilized monoclonal antibody. As a result of modulation of astrocytic function by gp120, the ensuing neuronal depolarization and glutamate exposure could activate both voltage-gated and N-methyl-D-aspartate-regulated Ca2+ channels, leading to increases in intraneuronal Ca2+ and neuronal death. These findings implicate the astrocyte directly in the pathogenesis of AIDS dementia complex.

Envelope glycoproteins from biologically diverse isolates of immunodeficiency viruses have widely different affinities for CD4.

The envelope glycoprotein gp120 of primate immunodeficiency viruses initiates viral attachment to CD4+ cells by binding to the CD4 antigen on host cell surfaces. However, among different CD4+ cell types, different viruses display distinct host cell ranges and cytopathicities. Determinants for both of these biological properties have been mapped to the env gene. We have quantitatively compared the CD4 binding affinities of gp120 proteins from viruses exhibiting different host cell tropisms and cytopathicities. The viral proteins were produced by using a Drosophila cell expression system and were purified to greater than 90% homogeneity. Drosophila-produced gp120 from T-cell tropic human immunodeficiency virus type 1 (HIV-1) BH10 exhibits binding to soluble recombinant CD4 (sCD4) and syncytia inhibition potency identical to that of pure authentic viral gp120. Relative to the affinity of HIV-1 BH10 gp120 for sCD4, that of dual tropic HIV-1 Ba-L is 6-fold lower, that of restricted T-cell tropic simian immunodeficiency virus mac is 70-fold lower, and that of noncytopathic HIV-2 ST is greater than 280-fold lower. Thus, viruses that utilize CD4 for infection do so by using a remarkably wide range of envelope affinities. These differences in affinity may play a role in determining cell tropism and cytopathicity.

Identification of the residues in human CD4 critical for the binding of HIV.

The CD4 molecule is a T cell surface glycoprotein that interacts with high affinity with the envelope glycoprotein of the human immunodeficiency virus, HIV, thus serving as a cellular receptor for this virus. To define the sites on CD4 essential for binding to gp120, we produced several truncated, soluble derivatives of CD4 and a series of 26 substitution mutants. Quantitative binding analyses with the truncated proteins demonstrate that the determinants for high affinity binding lie solely with the first 106 amino acids of CD4 (the V1 domain), a region having significant sequence homology to immunoglobulin variable regions. Analysis of the substitution mutants further defines a discrete binding site within this domain that overlaps a region structurally homologous to the second complementarity-determining region of antibody variable domains. Finally, we demonstrate that the inhibition of virus infection and virus-mediated cell fusion by soluble CD4 proteins depends on their association with gp120 at this binding site.

Identification of a potent, selective non-peptide CXCR2 antagonist that inhibits interleukin-8-induced neutrophil migration.

Interleukin-8 (IL-8) and closely related Glu-Leu-Arg (ELR) containing CXC chemokines, including growth-related oncogene (GRO)alpha, GRObeta, GROgamma, and epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), are potent neutrophil chemotactic and activating peptides, which are proposed to be major mediators of inflammation. IL-8 activates neutrophils by binding to two distinct seven-transmembrane (7-TMR) G-protein coupled receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB), while GROalpha, GRObeta, GROgamma, and ENA-78 bind to and activate only CXCR2. A chemical lead, which selectively inhibited CXCR2 was discovered by high throughput screening and chemically optimized. SB 225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea) is the first reported potent and selective non-peptide inhibitor of a chemokine receptor. It is an antagonist of 125I-IL-8 binding to CXCR2 with an IC50 = 22 nM. SB 225002 showed >150-fold selectivity over CXCR1 and four other 7-TMRs tested. In vitro, SB 225002 potently inhibited human and rabbit neutrophil chemotaxis induced by both IL-8 and GROalpha. In vivo, SB 225002 selectively blocked IL-8-induced neutrophil margination in rabbits. The present findings suggest that CXCR2 is responsible for neutrophil chemotaxis and margination induced by IL-8. This selective antagonist will be a useful tool compound to define the role of CXCR2 in inflammatory diseases where neutrophils play a major role.

Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4.

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcgamma receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.