Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors.

The present study was undertaken to further characterize the interaction of monoclonal antibodies (mAbs) against tumor necrosis factor (TNF) receptors with different targets, and to assess their ability to influence TNF effects on U937 and human endothelial cell (HEC) functions. Actions of recombinant TNF-alpha on U937 and HEC were effectively inhibited by Htr-5 and Utr-1, and to a greater extent by a combination of both mAbs. These observations indicate that TNF interaction with antigenically different components of membrane receptors (p55 and p75) represents a crucial step in transduction of signals for TNF toxicity against U937 and TNF activation of HEC functions.

Soluble CD14 participates in the response of cells to lipopolysaccharide.

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments.

Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment.

Elevated serum concentrations of activated hepatocyte growth factor activator in patients with multiple myeloma.

OBJECTIVES: Hepatocyte growth factor (HGF) is a potential key factor in multiple myeloma. Conversion of pro-HGF to its active form is a critical limiting step for its biological effects. We aimed to examine the levels of the most potent activator, the hepatocyte growth factor activator (HGFA), in serum and bone marrow plasma of patients with multiple myeloma. METHODS: The activated form of HGFA was measured by an enzyme-linked immunosorbent assay in serum (n = 49) and bone marrow plasma (n = 16) from multiple myeloma patients, and in serum from healthy controls (n = 24). RESULTS: The median concentrations of activated HGFA in myeloma and control sera were 39.7 (range 6.2-450.0) and 17.6 ng/mL (range 4.8-280.6), respectively. The difference was statistically significant (P = 0.037). The median concentration of activated HGFA in bone marrow plasma was 6.1 ng/mL (range 3.5-30.0). CONCLUSION: We here show for the first time that the activated form of HGFA is present at high levels in serum and bone marrow of myeloma patients, thus providing a necessary prerequisite for the activation of HGF.

Toll-like receptors mediate proliferation and survival of multiple myeloma cells.

Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in bone marrow (BM) and recurrent or persistent infections. Toll-like receptors (TLRs) are essential in the host defense against infections and today 10 human TLRs (TLR1-TLR10) and one TLR-homolog (RP105) have been characterized. B cells express several TLRs (mainly TLR1, 6, 7, 9, 10 and RP105) and TLR-initiated responses in B cells include proliferation, anti-apoptosis effect and plasma cell (PC) differentiation. The present study was designed to analyze the role of TLRs in MM. We show that frequent expressions of TLRs were detected in cell lines from MM patients (minimum six TLRs in each). In comparison, only few TLRs (mainly TLR1 and or RP105) were found expressed in PCs from BM of healthy donors. In addition, TLR-specific ligands induce increased proliferation and survival of the MM cell lines, partially due to an autocrine interleukin-6 production. Importantly, we demonstrate that also PC from MM patients proliferates in response to TLR-specific ligands. In conclusion, TLR-ligands may contribute to increased growth and survival of MM cells in MM patients.

Effect of malignant transformation, retinoic acid, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) on the sensitivity of rodent cells to Pseudomonas toxin.

A number of mouse and rat cells and their virus-transformed counterparts were tested for sensitivity to Pseudomonas aeruginosa exotoxin A (PEA). In each case, the transformed cells were considerably less sensitive than were the nontransformed cells. In the presence of trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, or retinoic acid, the transformed cells became as sensitive as the nontransformed cells, whereas these drugs had little or no effect on the sensitivity to PEA of the nontransformed cells. Temperature-sensitive virus-transformed normal rabbit kidney cells were sensitized to PEA by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, when these cells were grown as the transformed phenotype, whereas the nontransformed phenotype could not be sensitized. The possibility is discussed that upon malignant transformation a process which is dependent upon calmodulin or protein kinase C strongly decreases the sensitivity of the cells to PEA.

Intracellular glutathione determines bortezomib cytotoxicity in multiple myeloma cells.

Multiple myeloma (myeloma in short) is an incurable cancer of antibody-producing plasma cells that comprise 13% of all hematological malignancies. The proteasome inhibitor bortezomib has improved treatment significantly, but inherent and acquired resistance to the drug remains a problem. We here show that bortezomib-induced cytotoxicity was completely dampened when cells were supplemented with cysteine or its derivative, glutathione (GSH) in ANBL-6 and INA-6 myeloma cell lines. GSH is a major component of the antioxidative defense in eukaryotic cells. Increasing intracellular GSH levels fully abolished bortezomib-induced cytotoxicity and transcriptional changes. Elevated intracellular GSH levels blocked bortezomib-induced nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2)-associated stress responses, including upregulation of the xCT subunit of the Xc- cystine-glutamate antiporter. INA-6 cells conditioned to increasing bortezomib doses displayed reduced bortezomib sensitivity and elevated xCT levels. Inhibiting Xc- activity potentiated bortezomib-induced cytotoxicity in myeloma cell lines and primary cells, and re-established sensitivity to bortezomib in bortezomib-conditioned cells. We propose that intracellular GSH level is the main determinant of bortezomib-induced cytotoxicity in a subset of myeloma cells, and that combined targeting of the proteasome and the Xc- cystine-glutamate antiporter can circumvent bortezomib resistance.

Preparation and properties of a hybrid toxin of modeccin A-chain and ricin B-chain.

Hybrid molecules were prepared from the A- and B-chains of the two toxic lectins ricin and modeccin by dialyzing mixtures of isolated chains to allow a disulfide bridge to be formed between them. Whereas the hybrid consisting of ricin A-chain and modeccin B-chain was non-toxic, the converse hybrid, modeccin A-chain/ricin B-chain, was even more toxic to Vero cells than were the parent toxins, native ricin and modeccin. A number of drugs (NH4Cl, monensin, trifluoperazine, verapamil, ionophore A23187) which protect cells against modeccin, but not against ricin, protected to some extent against the toxic hybrid, but less so than against native modeccin. The possibility is discussed that the modeccin A-chain of the hybrid may enter the cytosol by two routes, one which is highly efficient and identical to that used by native modeccin and another less efficient one which cannot be used by native modeccin.

Evidence that membrane phospholipids and protein are required for binding of diphtheria toxin in Vero cells.

Treatment with phospholipase C strongly protected monkey kidney (Vero) cells against diphtheria toxin and reduced the ability of the cells to bind 125I-labelled toxin. Treatment with phospholipase D and with trypsin also protected the cells, although to a lesser extent. Phospholipase A2 had no protective effect. Phospholipase C also protected fetal hamster kidney cells against the toxin. After removal of the enzymes, as well as after treatment of the cells with 4-acetamide 4'-isothiocyanostilbene 2,2'-disulfonic acid, diphtheria toxin binding capability was restored slowly, apparently by a process requiring protein synthesis, since cycloheximide blocked the restoration. The data indicate that both phospholipids and protein are involved in the binding sites for diphtheria toxin.

Identification of new targets for therapy of osteolytic bone disease in multiple myeloma.

One of the most characteristic features of multiple myeloma is the development of osteolytic bone lesions. Myeloma-associated bone disease is caused by an increase in osteoclastic bone resorption and a decrease in osteoblastic new bone formation. Insight into the molecular mechanisms of osteoclastogenesis has been provided by the detection of receptor activator of NF-kappaB ligand (RANKL), its specific receptor (RANK) and its decoy receptor antagonist osteoprotegerin (OPG). The RANK signaling system is abnormally regulated in multiple myeloma and targeting this system may ameliorate myeloma bone disease. Less is known about the development of osteoblastic dysfunction, and further knowledge about the interaction between myeloma cells and osteoblasts is required. The aim of this review is to focus on the principles of bone biology for a better understanding of the development of myeloma bone disease and to identify possible therapeutic targets.

Differential expression of Toll-like receptor 2 in human cells.

Human Toll-like receptor 2 (TLR2) is a receptor for a variety of microbial products and mediates activation signals in cells of the innate immune system. We have investigated expression and regulation of the TLR2 protein in human blood cells and tissues by using two anti-TLR2 mAbs. Only myelomonocytic cell lines expressed surface TLR2. In tonsils, lymph nodes, and appendices, activated B-cells in germinal centers expressed TLR2. In human blood, CD14+ monocytes expressed the highest level of TLR2 followed by CD15+ granulocytes, and CD19+ B-cells, CD3+ T-cells, and CD56+ NK cells did not express TLR2. The level of TLR2 on monocytes was after 20 h up-regulated by LPS, GM-CSF, IL-1, and IL-10 and down-regulated by IL-4, IFN-gamma, and TNF. On purified granulocytes, LPS, GM-CSF, and TNF down-regulated, and IL-10 modestly increased TLR2 expression after 2 h. These data suggest that TLR2 protein expression in innate immune cells is differentially regulated by inflammatory mediators.

BRAF V600E mutation in early-stage multiple myeloma: good response to broad acting drugs and no relation to prognosis.

In this study, we analyzed the prevalence and clone size of BRAF V600E mutation in 209 patients with multiple myeloma and related the results to clinical phenotype, response and survival. Biopsies were screened for BRAF V600E by allele-specific real-time PCR (AS-PCR). Positive results were confirmed by immunohistochemistry, Sanger sequencing and, in three patients from whom we had stored purified myeloma cells, whole-exome sequencing. Eleven patients (5.3%) were BRAF V600E mutation positive by AS-PCR and at least one other method. The fraction of mutated cells varied from 4 to 100%. BRAF V600E-positive patients had no characteristic clinical phenotype except for significantly higher levels of serum creatinine (125 versus 86 µmol/l) Seven of eleven patients responded with at least very good partial response to alkylators, immunomodulatory agents or proteasome inhibitors. Progression-free and overall survival were similar in patients with and without the mutation. By this integrated approach, we found that patients with BRAF V600E mutation responded very well to broad acting drugs and there was no relation to prognosis in early-stage myeloma. In particular, a large mutated cell fraction did not correlate with aggressive disease.

Marked osteoblastopenia and reduced bone formation in a model of multiple myeloma bone disease in severe combined immunodeficiency mice.

We report on an in vivo model of human myeloma producing bone disease in irradiated severe combined immunodeficiency disease mice using the human myeloma cell line JJN-3 and its subline JJN-3 T1. The cell lines are not Epstein-Barr virus transformed and produce large amounts of hepatocyte growth factor (HGF). Mice had radiological signs of osteolysis and mild hypercalcemia. Xenografted cells were predominantly found in bone marrow and brown adipose tissue, but also in meninges and liver. Take was documented by histopathological examination, immunophenotyping of cultured bone marrow, and radiography. HGF was detected in serum and bone marrow plasma. Disease generally occurred within 45 days of intravenous inoculation and was signaled by paraparesis or signs of intracranial neoplasia. More than 90% of the mice had take of xenografts. The subline JJN-3 T1 gave more reproducible bone marrow take than the native cell line. Bone histomorphometric examination revealed a 99% reduction in osteoblast counts and a 33% reduction in osteoclast counts in areas of tumor growth. Bone formation rates were reduced by 53%. The results suggest that osteoblastopenia and reduced bone formation is of importance for the occurrence of osteolytic lesions in this model.

Entry mechanisms of protein toxins and picornaviruses.

The mode of entry into cells of a number of protein toxins with intracellular sites of action and of three picornaviruses is discussed. Of the different toxins in this group, diphtheria toxin has been most thoroughly studied with respect to its uptake mechanism. This toxin binds to cell surface receptors which are possibly part of the major anion-transport system in the cells. The bound toxin is then endocytosed and, when the pH drops below pH 5, a normally hidden hydrophobic domain is exposed and inserted into the membrane. By a process which, in addition to low pH, requires chloride transport and a proton gradient across the membrane, the toxin A fragment is translocated to the cytosol. When diphtheria toxin is bound at the cell surface, rapid entry through the surface membrane can be induced by treatment with low pH. Modeccin and Pseudomonas exotoxin A also require low pH for entry, but low pH is not able to induce rapid entry of these toxins from the cell surface. Another group of toxins, abrin, ricin and viscumin, is characterized by the fact that low pH in the medium prevents the toxins from entering the cytosol, but not from entering endocytic vesicles. However, when the pH is subsequently returned to neutrality the endocytosed toxins are able to enter the cytosol. In the picornaviruses the entry of a single hydrophilic macromolecule per cell is also sufficient to induce maximal biological effect. Poliovirus, like diphtheria toxin, appears to enter the cytosol from an acidic intracellular compartment which may be the endosome. Also human rhinovirus 2 requires low pH for entry, whereas encephalomyocarditis virus does not enter at low pH. The similarities and differences between the uptake mechanisms of toxins and viruses are discussed.

Hepatocyte growth factor reverses the TGF-beta-induced growth inhibition of CCL-64 cells. A novel bioassay for HGF and implications for the TGF-beta bioassay.

The influence of human hepatocyte growth factor (HGF) on the transforming growth factor beta (TGF-beta) bioassay CCL-64 was examined. HGF induced proliferation of the CCL-64 cells and potently counteracted TGF-beta-induced growth inhibition. HGF was not inactivated by transient acidification to pH 2, a commonly used procedure to activate latent TGF-beta. HGF was a stronger mitogen for the mink lung cells than epidermal growth factor (EGF), a known stimulator of CCL-64 cell growth. Costimulation of the cells by these two cytokines resulted in an additive effect on proliferation. In complex biological fluids containing large amounts of HGF, the TGF-beta concentration can be underestimated when determined by the CCL-64 assay. When a fixed amount of TGF-beta is added, the CCL-64 cells can be used as a reliable bioassay for HGF with a sensitivity of about 1 ng/ml.

The release of soluble p55 TNF receptor from U937 cells studied by a new p55 immunoassay.

A highly specific and sensitive immunoassay for soluble p55 tumor necrosis factor receptor (TNFR) has been established. The immunoassay was based on a newly developed monoclonal antibody (IV4E) recognizing a non-TNF-binding site of the p55 TNFR. The IV4E antibody immunoprecipitated a 55 kDa TNF binding protein from HL-60 cells. No binding of IV4E to the p75 TNFR could be detected. Bound TNFR to IV4E was detected with digoxigenin (DIG) labeled TNF. This assay could detect down to 300 pg/ml of soluble p55, which represents an 8-10-fold increase in sensitivity compared to earlier developed immunoassays. The assay was specific for soluble p55 TNFR present in serum and cell culture supernatants, since addition of excess unlabeled TNF together with DIG labeled TNF inhibited the signal. TNF concentrations up to 10 ng/ml in the TNFR sample did not affect the assay, indicating that TNFRs can be measured in samples containing TNF. The new immunoassay was used to study the mechanisms underlying the release of soluble p55 TNFR from U937 cells stimulated with TPA. The TPA induced release of soluble p55 TNFR from U937 cells occurred in two phases. First, a rapid increase of soluble p55 was observed after the addition of TPA. Later, the release of p55 occurred at a slower rate, and this release was inhibited by known inhibitors of protein synthesis and intracellular transport. Addition of TPA increased the p55 mRNA expression in U937 cells. The results suggest that TPA induces both release and new synthesis of p55 in U937 cells.

Development of immunoassays for the detection of soluble tumour necrosis factor receptors.

Immunoassays were established for the detection of the 55 kDa and 75 kDa tumour necrosis factor receptor (TNFR) fragments present in urine. The immunoassays were based on pairs of monoclonal TNFR antibodies directed against different epitopes of the 55 kDa and 75 kDa TNFRs. The immunoassays were judged to be specific for unoccupied TNFR since the signals were inhibited by adding recombinant human or murine TNF-alpha, and to a lesser extent by rTNF-beta (LT). Other cytokines such as IL-1 beta, IL-2 or rIFN-gamma did not affect the signal. In a preliminary screening it was found that urines from febrile patients contained higher amounts of 55 kDa and 75 kDa TNFR fragments than did urine from non-febrile individuals. The immunoassays could be used to monitor the purification of the two types of TNFR from the same febrile urine. Furthermore, the sensitivity and the speed of the assay could be increased by the use of magnetic beads as a solid support in the assay.

The role of hepatocyte growth factor and its receptor c-Met in multiple myeloma and other blood malignancies.

The cytokine hepatocyte growth factor (HGF) and its receptor c-Met are a ligand-receptor pair with important functions in a communicative interplay between HGF-producing, mesenchymal cells and c-Met-expressing target cells. HGF is cytoprotective and causes regeneration of parenchyma after tissue damage in several organs. The receptor c-Met was first characterized as an oncogene product being responsible for the transformation of an osteosarcoma cell line. HGF or c-Met is overexpressed in several human cancers, including various carcinomas. Some cells of hematopoietic origin also seem to be capable of c-Met expression, but the precise role of HGF in normal hematopoiesis is yet to be determined. In blood malignancies like acute myelogenous leukemia and, notably, multiple myeloma, HGF is overproduced and has implications for the prognosis of the patients. Biological significance of HGF overexpression in multiple myeloma is discussed and is likely to include effects on bone turnover and angiogenesis.

Elevated hepatocyte growth factor in sera from patients with insulin-dependent diabetes mellitus.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine known to be involved in tissue regeneration and repair. We measured serum levels of HGF in patients with insulin-dependent diabetes mellitus (type 1). The patients were divided into four groups: (1) 10 patients at clinical presentation before insulin treatment; (2) 19 patients with newly diagnosed type 1 diabetes (diabetes duration 1/2-3 years); (3) 14 patients with long-standing type 1 diabetes without renal involvement (diabetes duration >10 years, and urinary albumin excretion (UAER) <20 microg/ min); and (4) 20 patients with long-standing type I diabetes with renal involvement (diabetes duration >10 years and UAER 20-500 microg/min). Sera from 24 age- and sex-matched healthy blood donors constituted a control group. The HGF levels of the four groups were (mean +/- SD); group 1, 0.74+/-0.14; group 2, 0.78+/-0.40; group 3, 0.86+/-0.42; group 4, 0.79+/-0.27 ng/ml, compared to 0.43+/-0.24 ng/ml in the control group (P<0.0008). HGF levels were not significantly different between the four patient groups. The elevated serum HGF levels did not correlate with complications related to type 1 diabetes, such as UAER, retinopathy and macrovascular complications, suggesting that HGF levels were not associated with the type 1 diabetes complications. In conclusion, our results show that type 1 diabetic patients have increased serum HGF levels compared with controls and that HGF is elevated to the same extent in newly diagnosed as well as in long-standing type 1 diabetes.

Role of hepatocyte growth factor and its receptor c-met in multiple myeloma.

Multiple myeloma is characterised by the clonal expansion of malignant plasma cells. Recently, we reported that a new cytokine, hepatocyte growth factor (HGF), and its receptor c-met are related to this disease. Here we review the observations that associate HGF with myeloma. Malignant plasma cells produce HGF and express the receptor c-met. Many patients have elevated HGF levels, which is unfavourable both in terms of survival and response to treatment. Possible biological roles of HGF in this disease are discussed, with special focus on bone homeostasis and its binding to heparan sulphate proteoglycans.