Abstract. The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length eDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated eDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen eDNA. The central part of the Ki-67 antigen eDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycleassociated nuclear nonhistone proteins.
Unstimulated monocytes rapidly undergo physiological changes resulting in programmed cell death (apoptosis) while stimuli promoting differentiation of these cells into macrophages were shown to inhibit apoptotic processes. In the present study, we report that the platelet-derived -chemokine platelet factor 4 (PF4) induces the differentiation of monocytes into macrophages, as is evident from morphological changes as well as from the up-regulation of differentiation markers (carboxypeptidase M/MAX1 and CD71). Significant alterations of the phenotype were observed after 72 hours of culture in the presence of the chemokine and required a minimal concentration of 625 nmol/L PF4. PF4-induced macrophages were characterized by a lack of HLA-DR antigen on their surface but showed a strong increase in the expression of the CD28 ligand B7-2. Furthermore, PF4 stimulation prevented monocytes from undergoing spontaneous apoptosis during 72 hours of culture as determined in an annexin-V–binding assay. Although PF4 induced the secretion of relevant amounts of TNF-, neutralizing antibodies directed against TNF- or granulocyte-macrophage colony–stimulating factor (GM-CSF) did not revert PF4-induced rescue from programmed cell death, suggesting that PF4 exerts its antiapoptotic effects in a TNF-– or GM-CSF–independent fashion. On the basis of these results, we propose a novel role for PF4 in the control of monocyte differentiation during an inflammatory process in vivo.
Although platelet factor 4 (PF-4) and the beta-thromboglobulin (beta-TG) proteins represent the first chemokines to be discovered, their functional roles in host defense became clear only recently. Residing in platelets as storage proteins and becoming released into the blood at very high concentrations, these mediators appear to fulfill different and complementary tasks as first-line mediators in the recruitment and activation of leukocytes, as well in the regulation of tissue repair. Whereas both proteins are structurally closely related members of the CXC chemokine subfamily, they are subject to quite dissimilar regulatory mechanisms controlling their generation and their spectrum of biological activities. Thus, proteolytic processing of inactive precursors plays a decisive role in whether the beta-TG proteins will act as stimulatory or inhibitory agents in neutrophil activation via the G protein-coupled receptors CXCR-1 and 2. PF-4, existing as a single molecular form, is largely resistant to proteolytic modification, but its interaction with an unusual receptor(s) on leukocytes (a proteoglycan) appears to depend on its oligomeric state. There is growing evidence that both chemokines may interfere with each other at various regulatory levels to promote coordinated cell activation. Moreover, recent findings suggest novel and unexpected activities for these chemokines, which may extend our view on early host defense.
The clinical course of mycobacterial infections is linked to the capacity of pathogenic strains to modulate the initial antimycobacterial response of the macrophage. To elucidate some of the mechanisms involved, we studied early signal transduction events leading to cytokine formation by human monocyte-derived macrophages (MDM) in response to clinical isolates of Mycobacterium avium. TNF-α production induced by M. avium was inhibited by anti-CD14 mAbs, but not by Abs against the macrophage mannose receptor. Analysis of mitogen-activated protein (MAP) kinase activation (extracellular signal-regulated kinase 1/2, p38, and c-Jun NH2-terminal kinase) showed a rapid phosphorylation of all three subfamilies in response to M. avium, which was inhibited by anti-CD14 Abs. Using highly specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that activation of the extracellular signal-regulated kinase pathway, but not of p38, was essential for the M. avium-induced TNF-α formation. In contrast, IL-10 production was abrogated by the p38 inhibitor, but not by the MAP kinase kinase-1 inhibitor. In conclusion, M. avium-induced secretion of TNF-α and IL-10 by human macrophages is differentially regulated at the level of MAP kinase activity.
~,bstract Lipopolysaccharides (LPS, endotoxin) stimulate mononuclear cells to release cytokines which initiate endotoxic effects. Interaction of LPS at low concentrations with target cells is CDl4-dependent whereas at high LPS concentrations it is CD14-independent. Here, we demonstrate by resonance energy transfer (RET) technique that nonspecific, CDl4-independent intercalation of LPS into membrane systems can be mediated by lipopolysaccharide-binding protein (LBP). It is proposed that in this pathway, LBP breaks down LPS aggregates, transports the smaller units to and inserts them into the phospholipid cell matrix. We furthermore show that LBP also mediates the intercalation of other negatively charged amphiphilic molecules. We propose a model explaining CD14-independent cell activation at high endotoxin concentrations.
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