Simultaneous induction of HSP70 expression, and degranulation, in IgE/Ag-stimulated or extracellular HSP70-stimulated mast cells.

BACKGROUND: In mast cells, induction of HSP70 expression during antigen stimulation has not been reported. METHODS: Mouse bone marrow-derived mast cells (BMMC) were stimulated with IgE/Ag or HSP70. Induction of HSP70 expression and signaling protein phosphorylation were evaluated by immunoblotting. RESULTS: HSP70 expression is induced in BMMC at an early stage of IgE/Ag-dependent stimulation, some of which is released from the cells in a granule-associated form. Induction of HSP70 expression was also observed with an IgE/Ag-stimulated human basophilic cell line, indicating that the phenomenon is not restricted to mouse BMMC. The induction of HSP70 expression, and its release, followed a similar time course to that of degranulation. Released HSP70 seems to be responsible for degranulation and production of eicosanoids, at least in part, because a neutralizing anti-HSP70 antibody mitigated these activities and because exogenous HSP70 not only induced immediate degranulation followed by autocrine HSP70 expression but also enhanced degranulation in IgE/Ag-stimulated BMMC. Extracellular HSP70 was found to induce phosphorylation of linker for activation of T cells (LAT) and a series of downstream signaling molecules in BMMC. We further found that Fyn, Lyn, and spleen tyrosine kinase (Syk), which are known to concern LAT phosphorylation in IgE/Ag-stimulated BMMC, were not phosphorylated in HSP70-stimulated BMMC, whereas lymphocyte-specific protein tyrosine kinase (Lck) was phosphorylated. CONCLUSION: FcεRI stimulation in BMMC and basophils induces HSP70 expression and its release. Extracellular HSP70 induces degranulation and mediator release via phosphorylation of LAT.

Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor.

Idiopathic pulmonary alveolar proteinosis (I-PAP) is a rare disease of unknown etiology in which the alveoli fill with lipoproteinaceous material. We report here that I-PAP is an autoimmune disease with neutralizing antibody of immunoglobulin G isotype against granulocyte/macrophage colony-stimulating factor (GM-CSF). The antibody was found to be present in all specimens of bronchoalveolar lavage fluid obtained from 11 I-PAP patients but not in samples from 2 secondary PAP patients, 53 normal subjects, and 14 patients with other lung diseases. It specifically bound GM-CSF and neutralized bioactivity of the cytokine in vitro. The antibody was also found in sera from all I-PAP patients examined but not in sera from a secondary PAP patient or normal subjects, indicating that it exists systemically in I-PAP patients. As lack of GM-CSF signaling causes PAP in congenital cases and PAP-like disease in murine models, our findings strongly suggest that neutralization of GM-CSF bioactivity by the antibody causes dysfunction of alveolar macrophages, which results in reduced surfactant clearance.

Cellular interaction between fixed and living cells. Transfer of radioactive materials from living cells to fixed cells.

Transfer of radioactive materials to fixed cells from an overlying layer of living cells has been examined to determine whether fixed cells can act as acceptors of glycosyltransferases of living cells. After the incubation of living cells were removed by EDTA treatment, and the radioactivity associated with the fixed cells was determined. Lipids, proteins and carbohydrates were found to be transfered from the living cells to the fixed cells. The amount of radioactivity transferred to the fixed cells was dependent on the number of both fixed and living cells and increased with the time of incubation. When fixed cells were treated with chloroform-methanol before the addition of living cells, the transfer of both lipids and proteins to the fixed cells decreased drastically, but only a slight decrease incarbohydrate transfer was observed. Most of the radioactive materials transferred from living cells labeled with glucosamine or fucose to chloroform-methanol-treated fixed cells were solubilized by trypsin but not by the detergents tested. Approximately 55% of the materials transferred from the cells labeled with glucosamine could be solubilized by hyaluronidase and chondroitinase, and the rest was solubilized by neuraminidase and a glycosidase mixture. The treatment of chloroform-methanol-extracted fixed cells with trypsin caused a significant decrease in the transfer from cells labeled with glucosamine. When nucleotide sugars were used as the radioactive precursor, no significant amount of radioactivity was transferred to the fixed cells.

Expression of ecalectin, a novel eosinophil chemoattractant, in nasal polyps.

CONCLUSION: Ecalectin, which is produced in the mucosa of nasal polyps, seems to play an important role in the accumulation and activation of eosinophils in nasal polyps, regardless of the presence or absence of atopic predisposition. OBJECTIVE: Ecalectin is a recently discovered eosinophil chemoattractant which elongs to the galectin family. We investigated the expression of ecalectin in nasal polyp tissues associated with various nasal and paranasal diseases in order to clarify the pathogenesis of eosinophilia in nasal polyposis. MATERIAL AND METHODS: Nasal polyps were taken from 56 patients diagnosed as having chronic sinusitis with nasal polyposis. The surgically resected polyps and nasal turbinates were immunohistochemically stained using antibodies against EG2, human mast cell tryptase, CD3 and ecalectin. RESULTS: The number of EG2- and ecalectin-positive cells was significantly higher in nasal polyps than control turbinates. Ecalectin-positive cells were observed in the subepithelial layer, where many EG2-positive cells were present. The number of ecalectin-positive cells correlated significantly with the number of EG2-positive cells in nasal polyps. Many ecalectin mRNA-positive cells were also observed in nasal polyps with an accumulation of EG2-positive cells.

Inhibition of X-ray or chemical carcinogen-induced neoplastic transformation of C3H10T1/2 fibroblasts by lipopolysaccharides.

Oncogenic transformation of mouse 10T 1/2 fibroblasts induced upon exposure to X-ray or N-methyl-N'-nitro-N-nitrosoguanidine was suppressed if lipopolysaccharide (LPS) was present in the culture medium. The suppressive effect of LPS was exerted within 24 h after irradiation. Suppression was dependent on the concentration of LPS added and LPS (2 micrograms/ml) derived from Salmonella minnesota R595 reduced the number of transformed type III foci per dish from 0.39 to 0.15. Indomethacin (1 to 30 microM) further enhanced the effect of LPS in a dose-dependent manner.

Novel antibodies specific for proteolyzed forms of protein kinase C: production of anti-peptide antibodies available for in situ analysis of intracellular limited proteolysis.

We show here a novel method for the in situ analysis of proteolyzed proteins in a cell. As a model, we focused on protein kinase C (PKC) beta, which is cleaved at a specific site between the catalytic and regulatory domains by calpain, the intracellular calcium-activated neutral proteinase. To detect proteolyzed PKC beta 'cleavage-site-directed antibodies', which specifically recognize the amino-terminal region of the catalytic fragment but do not cross-react with the unproteolyzed enzymes, were raised using synthetic peptide. The synthetic peptide used in this study was QGTKVPEEKTT, corresponding to the amino-terminal region of the catalytic fragment from human PKC beta generated by calpain. Rabbits were immunized with the synthetic peptide after conjugation with a carrier protein. Antibodies obtained reacted with the 46-kDa catalytic fragment of PKC beta, whereas they did not cross-react with unproteolyzed enzyme nor other fragments with different amino-termini. Thus, our antibody is specific to the amino-terminal sequence QGTKVPEEKTT, but does not recognize the same sequence located internally in native PKC beta. When human monoblast U937 cells were treated with calcium ionophore, the catalytic fragment of PKC beta was detected in the cytosol by immunoblotting with the antibody. However, this antibody did not bind unproteolyzed 80-kDa PKC beta, although this form was dominant in the cytosol of the calcium ionophore-treated cells. We could also detect comparable amounts of catalytic fragment in the calcium ionophore-treated cells by immunocytochemical staining with the same antibody. Our method was applied to examine the proteolysis of PKC beta in neutrophils stimulated with various reagents.

Characterization of neutrophil b-type cytochrome in situ by electron paramagnetic resonance spectroscopy.

Electron paramagnetic resonance spectroscopy at 4.2 K was successfully used to characterize neutrophil b-type cytochrome in situ. The spectra of resting neutrophils taken under aerobic conditions gave a set of characteristic signals in a high magnetic field (g = 2.85, 2.21 and 1.67) beside signals for myeloperoxidase and others. From the g values, shapes and the results of other experiments, these signals were attributed to those of cytochrome b558. The results indicate that cytochrome b558 in resting neutrophils is a hexa-coordinated ferric hemoprotein in a low-spin state. The obtained gz and gx values for the hemichrome were consistent with that of bis(imidazole)-coordinated hemoprotein.

Chemotherapeutic activity of synthetic antimicrobial peptides: correlation between chemotherapeutic activity and neutrophil-activating activity.

The chemotherapeutic activity of three synthetic antibacterial peptides was investigated. KLKLLLLLKLK-NH2 and its D-enantiomer showed significant chemotherapeutic activity in MRSA-infected mice, whereas KLKLLLKLK-NH2, which showed the highest antibacterial activity among them in vitro, was found to have almost no ability to prevent MRSA infection. These results suggest that the antibacterial activity of peptides assessed in vitro does not necessarily correlate with their chemotherapeutic activity. We found that KLKLLLLLKLK-NH2 and its D-enantiomer, but not KLKLLLKLK-NH2, have the ability to activate human neutrophils to produce superoxide, suggesting that the prevention of MRSA infection by these peptides is not simply due to their direct bactericidal activity but to augmentation of the systemic defense mechanism mediated by neutrophils.

Lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte-macrophage colony stimulating factor.

Mice deficient in granulocyte-macrophage colony stimulating factor (GM-CSF) develop pulmonary alveolar proteinosis (PAP). We found that bronchoalveolar lavage fluid (BALF) from 11 patients with idiopathic pulmonary alveolar proteinosis (IPAP) suppressed the growth of peripheral blood monocytes and TF-1 cells, a cell line dependent on either GM-CSF or interleukin-3 (IL-3). The inhibitory effect of PAP-BALF occurred only when TF-1 cells were cultured with GM-CSF but not when cultured with IL-3, suggesting that PAP-BALF contains a factor that specifically interferes with GM-CSF function. 125I-GM-CSF binding to TF-1 cells was prevented in the presence of BALF from IPAP patients. Furthermore, cross-linking of 125I-GM-CSF to IPAP-BALF produced two major bands on SDS-PAGE; these bands were not observed in normal BALF. These data suggest that IPAP is caused by expression of binding factor(s) which inhibit GM-CSF function in the lung.

Significance of platelets in an antimetastatic activity of bacterial lipopolysaccharide.

Recently we reported an antimetastatic activity of bacterial lipopolysaccharide (LPS) on a NK-cell-resistant murine fibrosarcoma (NFSa). Here we investigate and report the mechanistic significance of platelets in this activity. The number of circulating platelets was reduced to 63% of the control 3 days after an i.v. injection of 1.0 micrograms LPS, and then recovered to the level of control at day 10. Aggregation efficiency of platelets was impaired by LPS. The number of metastatic lung colonies after an i.v. injection of tumor cells was maximally reduced to 2.2% of the control at day 3 and increased in proportion to the recovery of platelet number. Neuraminidase (Ndase), which caused a non-immunological thrombocytopenia, also inhibited lung metastasis when injected prior to an i.v. tumor cell challenge. LPS and Ndase showed an identical pattern against five other syngeneic tumors; these agents inhibited lung metastases of the FSa fibrosarcoma and the SCC VII squamous cell carcinoma but failed to inhibit those of the NR-S1 squamous cell carcinoma, the MMCa#4 mammary adenocarcinoma and the NR-PG parotid gland tumor. All the three cells which were not responsive to any agents possessed a high aggregating activity of platelets while the other three tumors responsive to both agents did not show a detectable level of this activity. Platelet transfusion failed to modify the antimetastatic activity of LPS. These results suggest that platelets play an important role in the antimetastatic activity of LPS, though whether the role is principal or assistant remains to be seen.

Antimetastatic activity of lipopolysaccharide against a NK-resistant murine fibrosarcoma.

An intravenous injection of bacterial lipopolysaccharide (LPS) into mice exerted prominent antimetastatic activities against a NK-resistant weakly immunogenic NFSa fibrosarcoma. The number of visible metastases in the lung was increased by a pretreatment of anti-asialoGM1 (asGM1) antibody or silica, but pretreatment of asGM1 antibody or silica scarcely affected the antimetastatic activities of LPS. The pulmonary retention of radiolabeled tumor cells showed that LPS accelerated the detachment of the tumor cells from the lung, and that this acceleration was not suppressed by anti-asGM1 antibody. Sialic acids of lung endothelial cell surface, essential components of various receptors, was diminished by the i.v. injection of LPS. These results suggested that the antimetastatic effect of LPS against NK-resistant NFSa cells was partly the result of modulations of lung endothelial cell surface.

Active components of intestinal bacteria for abdominal irradiation-induced inhibition of lung metastases.

We have previously reported that abdominal irradiation of mice inhibited lung metastases of a weakly immunogenic fibrosarcoma, and that transmigration after the irradiation of Enterobacter cloacae into mesenteric lymph nodes coincided with this phenomenon. In this paper, we show that Escherichia coli as well as E. cloacae reduce the number of metastatic lung colonies when these bacteria were intravenously injected into mice prior to the tumour cell challenge. The inhibition was caused not only by the administration of living bacteria but also by that of killed bacteria. Bacterial lipopolysaccharide (LPS), a component of membrane, replaced at least in part the effect of whole bacteria. Transfer of spleen cells from LPS-treated mice into intact recipients prominently inhibited metastatic development in the recipient mice. 'Cross transfer' between LPS high responders and LPS low responders suggested an indirect activity of transferred spleen cells. The antimetastatic activity of LPS depended on the tumour cell type; metastasis of fibrosarcomas was extensively inhibited by LPS irrespective of tumour immunogenicity while that of adenocarcinomas was only slightly inhibited. These results suggest that non-immunological mechanisms are involved in the antimetastatic activity of LPS.

Effects of diphtheria toxin and other exotoxins on oxidant generation by human and murine phagocytes.

Bacterial exotoxins such as diphtheria toxin (D.T.), staphylococcal alpha toxin and Leucocidin can powerfully activate granulocytes and macrophages as detected by production of chemiluminescence in presence of Luminol. Production of superoxide by granulocytes and of prostaglandin E2 in macrophages is also stimulated by D.T. In contrast with the known resistance of rodent parenchymal cells to the diphtheria toxin, human and rodent leucocytes have similar sensitivities to D.T.

Activation of human monocyte cell line U937 via cell surface calreticulin.

U937 cells were found to be activated by an antibacterial peptide, KLKLLLLLKLK-NH2 (L5), to generate superoxide anion (O2-)-like peripheral neutrophils. However, the state of cell surface calreticulin, a possible receptor for L5, was suggested to differ between neutrophils and U937 cells. Unlike the former, the latter ones were activated by anti-C-domain peptide antibody of calreticulin even in the absence of L5 and generated O2- in a GTP-binding protein (G-protein)-dependent manner.

Induction of CD40 in promyelocytic HL60 cells cultured with retinoic acid and/or various cytokines.

The antigenic protein CD40 on the surface of B lymphocytes plays an important role in their proliferation, immunoglobulin class switching, and rescue from apoptosis in the germinal center through interaction with T lymphocytes expressing CD40 ligand. The protein is also found on the cell surface of other antigen-presenting cells such as monocytes, dendritic cells, and thymic epithelium cells, but its presence in other myeloid cells has not been reported. We show here that CD40 protein is induced in promyelocytic HL60 cells, when cultured with retinoic acid, a vitamin that converts them to granulocyte-like cells. The cultured cells also expressed CD15, a marker for granulocytes, and cytochrome b(558), an essential component of the superoxide-generating system in phagocytes, on their surface. No detectable amount of mRNA for CD40 was found in naive HL60 cells, whereas a large amount of the message was induced in the cells cultured with the vitamin. Although CD40 expression was enhanced when the cells were further cultured with GM-CSF or IFN-gamma, expression of CD14, a marker for monocytes, was also enhanced. HL60 cells, therefore, express CD40 protein during differentiation not only toward monocytes but also toward granulocytes, at least transiently.

Erythropoietin 5'-flanking sequence-binding protein induced during hypoxia and cobalt exposure.

The 5'-flanking region of the human erythropoietin (Epo) gene contains a 0.14-kb sequence that is conserved in the Epo gene from mouse and located within a promoter that is activated under hypoxic conditions such as anemia. Using a fragment containing this sequence in DNA mobility shift assays, we found that specific DNA-binding proteins were induced in mouse kidney nuclei under anemic hypoxia. Using synthetic double-stranded oligonucleotides that contain this sequence, the essential binding site was defined to be the -40 to -20 region upstream of the transcription initiation site in the human Epo gene. By DNA affinity chromatography using a column with the immobilized 5'-flanking sequence, two inducible binding proteins with apparent molecular masses of 55 and 45 kDa were identified in the nuclei of mouse kidney and liver under anemic hypoxia. These binding proteins were also induced during cobalt exposure.

Reevaluation of the spin-trapped adduct formed from 5,5-dimethyl-1-pyrroline-1-oxide during the respiratory burst in neutrophils.

By employing EPR spectrometry with the aid of a spin-trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), the generation of superoxide anion and hydroxyl radical was reevaluated during the respiratory burst of porcine and human neutrophils. Properly prepared resting neutrophils did not generate any spin-trapped radical, and, when the cells were stimulated with phorbol myristate acetate, only DMPO-OOH, the spin-trapped adduct of superoxide anion, was detected. No formation of DMPO-OH, the spin-trapped adduct of the hydroxyl radical, was observed. DMPO-OOH was also detected principally when the neutrophils were stimulated with opsonized zymosan, a particulate stimulus. In the latter case, however, the formation of DMPO-OOH ceased shortly after the addition of zymosan and subsequent production of DMPO-OH was observed. The production of DMPO-OH was found to be associated with cell injury. DMPO at the concentration usually used for the experiment (0.045-0.09 M) injured phagocytizing neutrophils, causing lysis of the cells. On the other hand, an addition of cell homogenate or glutathione-glutathione peroxidase system to the suspension of intact cells which were producing DMPO-OOH resulted in the formation of DMPO-OH. Thus, DMPO-OH was probably derived from DMPO-OOH by the action of enzymes and/or factor(s) which were released from the lysed cells.

Suppression of superoxide-generating ability during differentiation of monocytes to dendritic cells.

Human peripheral monocytes cultured with GM-CSF and IL-4 differentiated to dendritic cells (DCs) and with GM-CSF alone to macrophages. Superoxide-generating ability in such DCs was found to be suppressed whereas that in macrophages remained constant. To examine the reason for the suppression in DCs, we evaluated by immunoblotting the levels of essential components of the superoxide generating system in the cells during the differentiation. In contrast to the levels of cytosolic 47- and 65-kDa components and Rac-p21, which remained constant throughout cultivation, those of the large and the small subunits of cytochrome b558 were found to decrease quickly by day 2 during cultivation of monocytes with GM-CSF and IL-4. DCs obtained after 7 days of cultivation had lost the large subunit almost completely and most of the small subunit. A cell surface epitope of the cytochrome detected by a monoclonal antibody also decreased during the differentiation. On the other hand, these components, including both subunits of cytochrome b558, were maintained in the cells during differentiation of monocytes to macrophages. These results indicate that the decreased levels of cytochrome b558, especially that of the large subunit, is responsible for the low level of superoxide-generating ability of DCs and that the suppression is caused by IL-4.

Effect of aromatic nitroso-compounds on superoxide-generating activity in neutrophils.

Aromatic nitroso-compounds such as nitrosobenzene inhibited the respiratory burst of intact neutrophils induced by various stimulants, including phorbol 12-myristate 13-acetate and a chemotactic peptide. The compounds also inhibited NADPH-dependent oxygen consumption by cell-free preparations of neutrophils. This indicates that nitroso-compounds act directly on the NADPH-oxidase system. The inhibitory effects induced by several nitroso-compounds, 2-nitrosotoluene, nitrosobenzene, 4-nitrosophenol, and 1-nitrosopyrrolidine, were examined and their inhibition constants, the concentrations causing 50% reduction of oxygen consumption, were found to be 0.043, 0.173, 0.672, and 32.1 mM, respectively. These values correlated well with the hydrophobicity of the compounds: a more hydrophobic compound was a more potent inhibitor against NADPH oxidase, suggesting that the oxidase has a hydrophobic site(s) for interaction with the inhibitors.

Induction of essential components of the superoxide generating system in human monoblastic leukemia U937 cells.

Cytochrome b558 composed of large and small subunits, and cytosolic 47- and 65-kDa proteins are important constituents of the superoxide (O2-) generating system in phagocytes and B lymphocytes. In this paper, we describe changes in O2(-)-generating activity and expression of O2(-)-generating components during differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. Undifferentiated U937 cells generated no O2- in response to a stimulation, although they expressed the three components other than the cytochrome b558 large subunit. When U937 cells were cultured with agents that induced the cell differentiation, such as vitamin D3, retinoic acid, interferon-gamma, and tumor necrosis factor, O2(-)-generating activity increased 5- to 200-fold depending on the agent used. Immunoblotting analysis revealed that the amounts of the four protein components essential for O2- generation increased, although their induction levels were significantly different between inducers. Among the four protein components, the cytochrome subunits were induced in low levels by all agents tested, which may explain why the O2(-)-generating activity of differentiated U937 cells was much lower than that of neutrophils from peripheral blood.