Eosinophil cationic protein alters proteoglycan metabolism in human lung fibroblast cultures.

Eosinophil cationic protein (ECP), a highly basic protein secreted from eosinophilic granulocytes, has been shown to take part in the inflammatory reaction. The involvement of ECP in fibroblast activation was therefore investigated in cell culture. Production of proteoglycans, hyaluronan and collagen in the presence of ECP was measured after incorporation of radioactive precursors and separation into different proteoglycan classes using gel and ion exchange chromatography and hydrophobic interaction chromatography. Proteoglycan accumulation in the cell layer was increased two- to fivefold at an ECP-concentration of 10 micrograms/ml. No effect on collagen, other proteins or hyaluronan was noted. Furthermore, no effect was observed on cell proliferation. The increased proteoglycan accumulation could be inhibited by addition of heparin or of antibodies to ECP. The effect could not be mimicked by the two basic peptides protamine and poly-L-lysine, speaking in favor of specificity. The increase in proteoglycan material was seen exclusively in the intracellular pool. No change of proteoglycans in the medium or the cell surface-associated pool was noted. The increase in the cell layer was accounted for by a two- to fivefold increase in free chains of heparan sulfate and dermatan sulfate. No change was seen in the proteoglycan pattern. No effect on proteoglycan synthesis or on endocytosis was noted. The increased accumulation of polysaccharide was caused by inhibited degradation of glycosaminoglycans. The half-lives of large and small heparan sulfate proteoglycans/glycosaminoglycans and dermatan sulfate proteoglycans/glycosaminoglycans in the cell layer are increased four- to sevenfold. We conclude that ECP inhibits proteoglycan degradation in fibroblasts, which indicates a role for the eosinophil in generation of fibrosis.     

Eosinophil cationic protein, specific IgE and IgG4 in human toxocariasis

Among 67 French patients presenting a toxocaral infection, various demographic, environmental, clinical and laboratory parameters (blood eosinophil count, eosinophil cationic protein (ECP), serum total IgE, specific IgE against common inhalant allergens, specific IgE and IgG4 against Toxocara excretory-secretory antigens) were investigated. Correlation studies and logistic regression analyses were conducted, testing elevated levels of ECP, specific anti-Toxocara IgE or IgG4 as outcome variables An elevated ECP level was significantly associated with both cough and rhinitis, a high level of specific anti-Toxocara IgE with itchy rashes and possible atopic status, and an increase of specific anti-Toxocara IgG4 with rural residence.     

Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases

Human eosinophil granules contain several basic proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and major basic protein (MBP). ECP and MBP are potent helminthotoxins while EDN is less so. Both ECP and EDN possess neurotoxic and ribonuclease activities. A clone representing ECP mRNA was isolated from an eosinophil lambda ZAP cDNA library. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids, the amino terminus of which is identical to the known partial amino acid sequence of ECP. The ECP nucleotide sequence shows similarity to EDN, rat pancreatic ribonuclease, and human angiogenin; all are members of the ribonuclease gene superfamily. Although the deduced amino acid sequence of ECP shares identical active site and substrate binding site residues with EDN, angiogenin, and human pancreatic ribonuclease, the ribonuclease activity of ECP is 50 to 100 times less than that of EDN possibly because of the lack of a positively charged residue at human pancreatic ribonuclease position 122. The calculated isoelectric point (10.8), electronic charge (14.5), and cationic charge distribution of ECP are different from those of EDN but similar to those of MBP, which may account in part for the greater helminthotoxic activity of ECP when compared to EDN. These data suggest that ECP and EDN are derived from a common ancestral ribonuclease gene and that ECP has evolved into a potent helminthotoxin similar in some respects to MBP, while losing much of its ribonuclease activity.     

Rapamycin inhibits proliferation and differentiation of human endothelial progenitor cells in vitro

Bone-marrow-derived, circulating endothelial precursor cells contribute to neoangiogenesis in various diseases. Rapamycin has recently been shown to have anti-angiogenic effects in an experimental tumor model. Our group has developed a culture system that allows expansion and endothelial differentiation of human CD133(+) precursor cells. We could show by PCR analysis that mTOR, the rapamycin-binding protein, was expressed in fresh CD133(+) cells, in expanded cells after 28 days, and in differentiated endothelial cells. Rapamycin inhibited proliferation of CD133(+) cells dose dependently at similar concentrations as hematopoietic Jurkat or HL-60 cells. Apoptosis was induced by rapamycin after 48 h of treatment, which could be reduced by preincubation with FK 506. Furthermore, the development of adherent endothelial cells from expanded CD133(+) cells was dose dependently inhibited. Expression of endothelial antigens CD144 and von Willebrand factor on differentiating endothelial precursors was reduced by rapamycin. In summary, rapamycin inhibits proliferation and differentiation of human endothelial precursor cells underlining its anti-angiogenic effects.     

IL-10 inhibits human T cell proliferation and IL-2 production.

Human IL-10 has been reported previously to inhibit the secretion of IFN-gamma in PBMC. In this study, we have found that human IL-10 inhibits T cell proliferation to either mitogen or anti-CD3 mAb in the presence of accessory cells. Inhibited T cell growth by IL-10 was associated with reduced production of IFN-gamma and IL-2. Studies of T cell subset inhibition by human IL-10 showed that CD4+, CD8+, CD45RA high, and CD45RA low cells are all growth inhibited to a similar degree. Dose response experiments demonstrated that IL-10 inhibits secretion of IFN-gamma more readily than T cell proliferation to mitogen. In addition, IL-2 and IL-4 added exogenously to IL-10 suppressed T cell cultures reversed completely the inhibition of T cell proliferation, but had little or no effect on inhibition of IFN-gamma production. Thus, in addition to its previously reported biologic properties, IL-10 inhibits human T cell proliferation and IL-2 production in response to mitogen. Inhibition of IFN-gamma production by IL-10 appears to be independent of the cytokine effect of IL-2 production.     

Eosinophil cationic protein high-affinity binding to bacteria-wall lipopolysaccharides and peptidoglycans

The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. The protein displays antimicrobial activity against both Gram-negative and Gram-positive strains. The protein can destabilize lipid bilayers, although the action at the membrane level can only partially account for its bactericidal activity. We have now shown that ECP can bind with high affinity to the bacteria-wall components. We have analyzed its specific association to lipopolysaccharides (LPSs), its lipid A component, and peptidoglycans (PGNs). ECP high-affinity binding capacity to LPSs and lipid A has been analyzed by a fluorescent displacement assay, and the corresponding dissociation constants were calculated using the protein labeled with a fluorophor. The protein also binds in vivo to bacteria cells. Ultrastructural analysis of cell bacteria wall and morphology have been visualized by scanning and transmission electron microscopy in both Escherichia coli and Staphylococcus aureus strains. The protein damages the bacteria surface and induces the cell population aggregation on E. coli cultures. Although both bacteria strain cells retain their shape and no cell lysis is patent, the protein can induce in E. coli the outer membrane detachment. ECP also activates the cytoplasmic membrane depolarization in both strains. Moreover, the depolarization activity on E. coli does not require any pretreatment to overcome the outer membrane barrier. The protein binding to the bacteria-wall surface would represent a first encounter step key in its antimicrobial mechanism of action.     

Interferon gamma inhibits proliferation and hyaluronic acid adhesion of human malignant glioma cells in vitro

Malignant gliomas are frequent and the prognosis is poor. The cytokine interferon gamma (IFN-gamma) enhances several immune phenomena and may be used in immunotherapy of tumours. Therefore we investigated the influence of IFN-gamma on human cell lines T98G, U87MG, 86HG39 and 85HG66, measuring cell viability (MTT-test) and proliferation (3H-thymidine uptake). IFN-gamma markedly decreased viability and proliferation of all investigated cell lines. Expression of CD44 and adhesion to hyaluronic acid (HA) are involved in glioma invasion. Influence of IFN-gamma on these two features has also been investigated. IFN-gamma markedly decreased HA-adhesion in all three investigated cell lines, whereas CD44 expression remained uninfluenced. To summarise, IFN-gamma strongly decreased cell growth and HA-adhesion of malignant glioma cell lines in vitro. We suggest further investigations to characterise better the role of IFN-gamma as a treatment opportunity for malignant gliomas.     

Eosinophil cationic protein: overview of biological and genetic features

The eosinophil cationic protein (ECP) is a small polypeptide that originates from activated eosinophil granulocytes. A wide range of stimuli has been shown to induce the secretion of ECP. The gene that encodes the human ECP is located on chromosome 14, and the protein shares the overall three-dimensional structure and the RNase active-site residues with other proteins in the RNase A superfamily. Several single-nucleotide polymorphisms in the human ECP gene have been currently described. ECP has many biological functions, including an immunoregulatory function, the regulation of fibroblast activity, and the induction of mucus secretion in the airway. Additionally, the protein is a potent cytotoxic molecule and has the capacity to kill mammalian and nonmammalian cells. The purpose of this article was to review the known biological and genetic characteristics of ECP that contribute to the understanding of this protein's role in the development and progression of a wide variety of diseases.     

Adherence of human erythroleukemia cells inhibits proliferation without inducing differentiation.

To investigate the effect of extracellular matrix molecules in the megakaryocytic lineage, we studied the role of integrin engagement in the proliferation and differentiation of human erythroleukemia (HEL) cells. HEL cells grew in suspension, but their adherence depended upon the presence of matrix proteins or protein kinase C signaling. Adherence by itself did not trigger commitment of these cells but accelerated phorbol 12-myristate 13-acetate-induced differentiation. HEL cells adhered to fibronectin mainly through alpha5beta1, and this receptor acted synergetically with alpha4beta1. Integrin engagement induced cell growth arrest through mitogen-activated protein kinase inactivation. Such down-regulation of the mitogen-activated protein kinase pathway by integrin engagement was suggested as a megakaryocytic-platelet lineage specificity. This signaling was not restricted to a peculiar integrin but was proposed as a general mechanism in these cells.     

The effect of 1,25-dihydroxyvitamin D3 on in vitro immunoglobulin production in human B cells

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently suppressed immunoglobulin (Ig) production of human B cells, as evaluated by IgG-plaque-forming cells (IgG-PFC) in the culture of pokeweed mitogen (PWM)-activated B cells. Similar suppressive effect of 1,25(OH)2D3 on Ig production of B cells was observed in the Staphylococcus aureus Cowan I(SAC)-induced Ig-producing system. The mean percentage of inhibitions at a concentration of 10(-9) M were 60.0 +/- 8.2% (mean +/- SE, n = 6) and 65.1 +/- 4.7% (n = 10) in PWM- and SAC-stimulated cultures, respectively. The suppression was strongly exhibited only when 1,25(OH)2D3 was added at the start of the 6-day culture, accompanied by a decrease in DNA synthesis of B cells in both culture systems. On the other hand, the addition of 1,25(OH)2D3 on day 4, when DNA synthesis reached at plateau and IgG-PFC began to be detectable, had no noticeable affect on either the number of PFC or DNA synthesis of B cells. Furthermore, 1,25(OH)2D3 suppressed Ig production even when B cells were exposed to the agent for 4 hr after the activation with PWM or SAC, but not before the activation. These results indicate that 1,25(OH)2D3 inhibits B cell proliferation before differentiation to Ig-secreting cells, consequently reducing Ig production; and that its action appears to be mediated by the cytosol receptors expressed on activated B cells. Thus, the agent may serve as an immunoregulating hormone in vivo, as well as in vitro.     

1-Oleoyl-2-acetylglycerol promotes immunoglobulin production independent of cell proliferation in human peripheral blood mononuclear cells

1-Oleoyl-2-acetylglycerol (OAG) stimulated IgG and IgM production in a dose-dependent manner in human peripheral blood mononuclear cells (PBM) but not PBM proliferation. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) did not stimulate Ig production. OAG did not stimulate an increase in IL-2 generation or IL-2 receptor expression. H-7, a protein kinase C blocker completely inhibited OAG-stimulated Ig production. The results suggest that OAG stimulation of Ig production is independent of cell proliferation; a generalized increase in T-cell activation does not appear to be necessary in the OAG stimulation of Ig production. Finally, PBMs respond differently to OAG and TPA although both are protein kinase C activators.     

Identification of the human plasma protein which inhibits fibrinolysis associated with malignant cells

Mixed cultures of mouse fibroblasts and mouse fibroblasts transformed with Kirsten murine sarcoma virus were grown in petri dishes and overlayed with casein. The appearance of focal lysis zones required the presence of transformed cells in the culture and plasminogen in the overlay, indicating that caseinolysis was due to plasminogen activator released by the malignant cells. Caseinolysis was inhibited by addition of human plasma or bovine pancreatic trypsin inhibitor to the overlay, 1 ml of plasma being equivalent to 67 ± 18 (mean ± S.E.) kallikrein inhibitor (KI) units of trypsin inhibitor.The culture fluid of a human melanoma line induced lysis of a fibrin clot, 1 ml of culture fluid being equivalent to 250 CTA units of urokinase (EC 3.4.99.26). Fibrinolysis was inhibited by addition of human plasma or trypsin inhibitor, 1 ml of plasma being equivalent to 94 ± 34 KI units of trypsin inhibitor.Specific removal of antiplasmin, the fast-reacting plasmin inhibitor (Collen, D. (1976) Eur. J. Biochem. 69, 209), from plasma by immunoabsorption completely abolished its inhibitory activity, both in the caseinolytic and fibrinolytic assays. It is therefore concluded that antiplasmin is the only protein in human plasma capable of inhibiting the fibrinolytic activity associated with oncogenic transformation or neoplasia. Whether this effect is exclusively due to inhibition of formed plasmin or also to interference with plasminogen activvtion remains unsettled.     

TGF-beta inhibits proliferation of and promotes differentiation of human promonocytic leukemia cells.

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in a variety of responses associated with wound healing and inflammation. Thus, TGF-beta 1 enhances production of several extracellular matrix proteins both in vitro and in vivo, is chemotactic for monocytes, and alters the functioning of lymphocytes. We have examined the ability of TGF-beta 1 to affect the behavior of human THP-1 promonocytic leukemia cells, a cell line with the capacity to differentiate into macrophage-like cells. TGF-beta 1 reduces the growth rate of these cells, induces morphologic changes, and promotes adherence to culture surfaces. In addition, the adherent cell population expresses high levels of esterase activity, acquires the ability to ingest latex beads, and releases elevated levels of interleukin 1. TGF-beta 1-treated cells also express elevated levels of the beta 2 family of integrins. Taken together, these results suggest that TGF-beta 1 is capable of promoting the maturation of promonocytic cells into macrophages. This outcome has implications at wound sites where TGF-beta 1 and a myriad of other factors interact with many cell types to facilitate healing.     

Involvement of cationic channels in proliferation and migration of human mesenchymal stem cells

Human mesenchymal stem cells (HMSCs) have been applied in various clinic settings. Ion channels play an important role in cellular physiology. However, the potential role of cationic channels in regulating the proliferation and migration properties of hMSCs remains to be determined. In the present study, the functional expression of ion channels in hMSCs was investigated by patch clamp. MTT assay and BrdU stainings were used to assess the proliferation of hMSCs. hMSC migration was evaluated by Transwell migration assays. The results show that sodium-, L-type calcium, potassium currents have been identified in hMSCs. TEA (K⁺ channel blocker), nifedipine (Ca²⁺ channel blocker) can inhibit both proliferation and migration of hMSCs. The increase of extracellular Ca²⁺ concentration promoted both proliferation and migration of hMSCs. TTX, a Na⁺ channel blocker, promoted cell proliferation but inhibited cell migration. Our data suggest that cationic channels (sodium, L-type calcium, potassium channels) play important roles in regulating proliferation and migration of hMSCs.     

PPD-induced immunoglobulin production in human peripheral blood lymphocytes. II. Separation of PPD-reactive helper T cells from PWM-reactive helper T cells in polyclonal immunoglobulin production of human peripheral blood lymphocytes.

We investigated the relationship bewteen PPD-reactive helper T cells and PWM-reactive helper T cells in polyclonal Ig production of human PBL. Elimination of PPD-reactive T cells by BUdR + light treatment resulted in a loss of helper function in PPD-induced Ig production, but had no effect on helper function in PWM-induced Ig production. On the other hand, elimination of PWM-reactive T cells resulted in a loss of helper function in both PPD-induced Ig production and PWM-induced Ig production. A blast cell-enriched fraction that was generated by PPD and separated by the velocity sedimentation method contained helper function in both responses. On the other hand, blast cell-depleted fractions did not contain PPD-reactive helper function, although the PWM-reactive helper function was evident. These results strongly suggest that PPD-reactive helper T cells are included in PWM-reactive helper T cells.