Interaction of C1q receptor with lung surfactant protein A.

Earlier we reported the purification of C1q receptor (C1qR) from U937 cells and human tonsil lymphocytes (Malhotra, R. and Sim, R. B., Biochem. J. 1989. 218: 625) and showed that C1qR interacts with the ligands C1q, mannose-binding protein, conglutinin and lung surfactant protein A (SP-A) (Malhotra, R., Thiel, S., Reid, K. B. M. and Sim, R. B., J. Exp. Med. 1990. 172: 955). C1qR was characterized as an acidic glycoprotein, which, when solubilized, exists as a dimer of Mr 115,000 under non-denaturing conditions. In this article we provide evidence for binding of radioiodinated SP-A to U937 cells and show that binding of radioiodinated SP-A to U937 cells is specific, saturable, salt dependent and is inhibited by purified C1qR and by C1q. The interaction of SP-A with U937 cells was found to up-regulate the surface expression of C1qR. Incubation of SP-A with U937 cells at 37 degrees C for 80 min was found to increase the receptor number per cell. Increase in receptor number was inhibited in the presence of sodium azide and monensin. Incubation of cells with calcium ionophore A23187 induced increased surface expression in the absence of SP-A. The results indicate that interaction of SP-A with U937 cells triggers the expression of an intracellular pool of C1qR.     

Progressive lung disease and surfactant dysfunction with a deletion in surfactant protein C gene

Mutations in the surfactant protein (SP)-C gene are responsible for familial and sporadic interstitial lung disease (ILD). The consequences of such mutations on pulmonary surfactant composition and function are poorly understood. To determine the effects of a mutation in the SP-C gene on surfactant, we obtained lung tissue at the time of transplantation from a 14-mo-old infant with progressive ILD. An in-frame 9-bp deletion spanning codons 91-93 in Exon 3 of the SP-C gene was present on one allele; neither parent carried this deletion. SP-C mRNA was present in normal size and amount. By immunofluorescence, proSP-C was aggregated within alveolar Type II cells in a compartment separate from SP-B. In airway surfactant, there was little or no mature SP-B or SP-C; SP-A content was increased. Minimum surface tension was increased (20 mN/m, normal < 5 mN/m). Type II cells contained normal and disorganized appearing lamellar bodies by electron microscopy. This spontaneous deletion on one allele of the SP-C gene was associated with sporadic ILD and abnormalities in surfactant composition and function. We propose that a dominant negative effect on surfactant protein metabolism and function results from aggregation of misfolded proSP-C and subsequent cell injury and inflammation.     

Clinical and ultrastructural spectrum of diffuse lung disease associated with surfactant protein C mutations

Genetic defects of surfactant metabolism are associated with a broad range of clinical manifestations, from neonatal respiratory distress syndrome to adult interstitial lung disease. Early therapies may improve symptoms but diagnosis is often delayed owing to phenotype and genotype variability. Our objective was to characterize the cellular/ultrastructural correlates of surfactant protein C (SP-C) mutations in children with idiopathic diffuse lung diseases. We sequenced SFTPC – the gene encoding SP-C – SFTPB and ABCA3, and analyzed morphology, ultrastructure and SP expression in lung tissue when available. We identified eight subjects who were heterozygous for SP-C mutations. Median age at onset and clinical course were variable. None of the mutations were located in the mature peptide-encoding region, but were either in the pro-protein BRICHOS or linker C-terminal domains. Although lung morphology was similar to other genetic surfactant metabolism disorders, electron microscopy studies showed specific anomalies, suggesting surfactant homeostasis disruption, plus trafficking defects in the four subjects with linker domain mutation and protein misfolding in the single BRICHOS mutation carrier in whom material was available. Immunolabeling studies showed increased proSP-C staining in all cases. In two cases, amyloid deposits could be identified. Immunochemistry and ultrastructural studies may be useful for diagnostic purposes and for genotype interpretation.     

A monoclonal antibody (HO-No-1) with specificity for a human nucleolar protein.

A murine monoclonal antibody (named HO-No-1) which specifically reacts with a target antigen of 72,000 mol. wt (p72) in the nucleoli of human cells, has been isolated. Using an avidin-biotin peroxidase immunoperoxidase method, it was found that this antibody stains exclusively the nucleolus and not other portions of the nucleus and cytoplasm. This reaction pattern was observed consistently, although to different degrees in 14 human normal tissues and human malignant cell lines. However, after pretreatment with actinomycin D (250 ng/ml) for 2 h, the antibody stains the nucleoplasm uniformly. Thus this study demonstrates evidence for an antibody which reacts specifically with human nucleoli to a protein which could play an important role in rRNA synthesis.     

An improved method for the detection of IgE antibody of defined specificity by ELISA using rat monoclonal anti-IgE antibody

An improved method for the detection of IgE antibody of defined specificity by ELISA using rat monoclonal anti-IgE antibodies is described. The innovation consists of coating the plates first by a monoclonal rat anti-murine IgE antibody, adding the sera to this antibody-coated plates and then adding the biotin-conjugated antigen after the sera. The plates are then reacted with streptavidin-peroxidase and developed. This procedure eliminates possible competitions with other isotypes of the same specificity. The method is useful especially to quantitate IgE with defined specificity in the presence of high amounts of isotypes of the same specificity.     

慢性阻塞性肺疾病大鼠肺组织中肺表面活性物质蛋白-C表达下降

目的探讨肺表面活性物质蛋白-C(SP-C)在慢性阻塞性肺疾病(COPD)大鼠肺组织中的作用。方法将大鼠随机分为对照组、香烟烟雾暴露组、脂多糖组和COPD组,每组10只。测定各组大鼠的PaO_2和PaCO_2的水平;透射电镜观察肺组织的细胞微观结构;ELISA检测支气管肺泡灌洗液(BALF)和肺组织SP-C蛋白;RT-q PCR检测肺组织SP-C mRNA的表达。结果与其他组相比,COPD大鼠的PaO_2最低,而PaCO_2最高;肺泡Ⅱ型上皮细胞表面微绒毛明显减少(P<0.01);BALF和肺组织中SP-C蛋白表达下降(P<0.01);肺组织中的SP-C mRNA表达下降(P<0.01)。结论 SP-C在COPD大鼠肺组织中表达下调,这种下调可能引起肺通气和肺换气功能障碍。     

The specificity of antibody formation in mice following immunization with hapten-carrier complexes mixed with the surfactant, dimethyl dioctadecyl ammonium bromide.

Mice were immunized i.c. with various enlarged haptens conjugated to bovine serum albumin and mixed with the cationic, surface active lipid, dimethyl dioctadecyl ammonium bromide (DDA). This immunization generated delayed-type hypersensitivity (DH) in these mice without detectable concomitant antibody formation. The DH was measured as footpad swelling. However, both direct and indirect hapten-specific PFC could be detected in peripheral lymph nodes and in spleens 4 days after a challenge injection. Although the adjuvant, DDA, promotes a strong cross-reactivity in DH between heterologous hapten-carrier complexes, the antibody-forming cells produced 4 days after challenge showed relatively high specificity for the immunizing hapten. This indicates only weak cross-reactivity at the antibody-forming cell level co-existent with high cross-reactivity in DH expression to the same hapten-carrier complexes. These results are consistent with the possibility that B-cell receptors may be capable of expressing a greater degree of hapten specificity than T-cell receptors. It is tentatively concluded that Thelper cells participating in antibody formation may represent a subset of the T cells involved in DH.     

Complexing of bacterial lipopolysaccharide with lung surfactant.

Lipopolysaccharides (LPS) from Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Serratia marcescens, or Pseudomonas aeruginosa were mixed with pulmonary surfactant to investigate their in vitro interaction. After 6 h of incubation at 37 degrees C, LPS-surfactant mixtures were examined by sucrose density gradient centrifugation. The E. coli LPS-surfactant mixture was examined by immunoelectron microscopy with protein A-colloidal gold. The binding that occurred between LPS and the surfactant vesicles resulted in a complex with a density higher than the density of the surfactant alone. The protein A-colloidal gold identified LPS in the LPS-surfactant complexes. The toxicity of E. coli LPS was enhanced by complexing with the surfactant when compared with the intraperitoneal injection into CF1 mice, even at a 64:1 ratio of surfactant to LPS. The complexing of LPS and surfactant in the lung may alter the physiologic properties of surfactant that contribute to the physiopathological changes observed with some types of pneumonia.     

Erythropoietin receptor expression in non-small cell lung carcinoma: a question of antibody specificity

Immunohistochemical studies on formalin-fixed, paraffin-embedded (FFPE) tissue utilizing polyclonal antibodies form the cornerstone of many reports claiming to demonstrate erythropoietin receptor (EPOR) expression in malignant tissue. Recently, Elliott et al. (Blood 2006;107:1892-1895) reported that the antibodies commonly used to detect EPOR expression also detect non-EPOR proteins, and that their binding to EPOR was severely abrogated by two synthetic peptides based on the sequence of heat shock protein (HSP) 70, HSP70-2, and HSP70-5. We have investigated the specificity of the C20 antibody for detecting EPOR expression in non-small cell lung carcinoma (NSCLC) utilizing tissue microarrays. A total of 34 cases were available for study. Antibody absorbed with peptide resulted in marked suppression of cytoplasmic staining compared with nonabsorbed antibody. Four tumors that initially showed a membranous pattern of staining retained this pattern with absorbed antibody. Positive membranous immunoreactivity was also observed in 6 of 30 tumors that originally showed a predominantly cytoplasmic pattern of staining. Using the C20 antibody for Western blots, we detected three main bands, at 100, 66, and 59 kDa. Preincubation with either peptide caused abolition of the 66-kDa band, which contains non-EPOR sequences including heat shock peptides. These results call into question the significance of previous immunohistochemical studies of EPOR expression in malignancy and emphasize the need for more specific anti-EPOR antibodies to define the true extent of EPOR expression in neoplastic tissue.     

Hormonal effects on the surfactant protein B (SP-B) mRNA in cultured fetal rat lung

Glucocorticoids, triiodothyronine (T3), and cyclic adenosine monophosphate (cAMP) have been shown previously to modulate phosphatidylcholine and surfactant protein A (SP-A) synthesis in fetal rat lung explant cultures. In this report, we have examined the hormonal regulation of the rat surfactant protein B (SP-B) mRNA to determine whether SP-B expression is coordinately regulated with the surfactant phospholipids or with SP-A. Dexamethasone (1 to 200 nM) and cAMP (200 microM) had a stimulatory effect on SP-B mRNA levels, whereas T3 tended to inhibit the accumulation of SP-B mRNA. In combination experiments, treatment with dibutyryl-cAMP (200 microM) and dexamethasone (100 nM) resulted in about a 22-fold increase, whereas dexamethasone or dibutyryl-cAMP alone produced 18- and 2-fold increases, respectively. When the cAMP analogue 8-bromo-cAMP (200 microM) was used in combination with dexamethasone, there was no significant difference between the combined effect and that of dexamethasone alone. T3 treatment, however, resulted in a significant reduction of the dexamethasone-induced stimulation from about a 22-fold to a 14-fold increase. Tissue in situ hybridization showed that dexamethasone stimulated the levels of SP-B mRNA in cells from both the alveolar and bronchiolar epithelium. These data indicate that there are differences in the hormonal regulation of the components of surfactant, suggesting that they are independently regulated.     

A monoclonal antibody (HO-No-1) with specificity for a human nucleolar protein

A murine monoclonal antibody (named HO-No-1) which specifically reacts with a target antigen of 72,000 mol. wt (p72) in the nucleoli of human cells, has been isolated. Using an avidin-biotin peroxidase immunoperoxidase method, it was found that this antibody stains exclusively the nucleolus and not other portions of the nucleus and cytoplasm. This reaction pattern was observed consistently, although to different degrees in 14 human normal tissues and human malignant cell lines. However, after pretreatment with actinomycin D (250 ng/ml) for 2 h, the antibody stains the nucleoplasm uniformly. Thus this study demonstrates evidence for an antibody which reacts specifically with human nucleoli to a protein which could play an important role in rRNA synthesis.     

Interaction of pulmonary surfactant protein C with CD14 and lipopolysaccharide

In addition to their effects on alveolar surface tension, some components of lung surfactant also have immunological functions. We found recently that the hydrophobic lung surfactant protein SP-C specifically binds to the lipid A region of lipopolysaccharide (LPS). In this study, we show that SP-C also interacts with CD14. Four observations showed cross talk between the three molecules SP-C, LPS, and CD14. (i) Like LBP, SP-C allows the binding of a fluorescent LPS to cells expressing CD14 (the other surfactant components were ineffective). (ii) Recombinant radiolabeled CD14 and SP-C (or a synthetic analog of SP-C) interact in a dose-dependent manner. (iii) LPS blocks the binding of radiolabeled CD14 to SP-C-coated wells. (iv) SP-C enhances the binding of radiolabeled CD14 to LPS-coated wells. These results, obtained with native murine SP-C and with three synthetic analogs, suggest that LPS and CD14 interact with the same region of SP-C and that binding of SP-C modifies the conformation of CD14 or the accessibility of its LPS-binding site, allowing it to bind LPS. This ability of SP-C to interact with the pattern recognition molecule CD14 extends the possible immunological targets of SP-C to a large panel of microorganisms that can enter the airways.