Marja-Leena Majuri

Recombinant E-selectin-protein mediates tumor cell adhesion via sialyl-Lea and sialyl-Lex

E-selectin (previously known as ELAM-1) is one of the adhesion molecules expressed on activated endothelium. Here we show that HL-60 cells express sialyl-Le(x), but not Sialyl-Le(a) on their surface, a colon carcinoma cell line COLO 205 express both these epitopes and another colon carcinoma COLO 320 does not express either one of them. HL-60 and COLO 205 cell adhere strongly to E-selectin coated microwells, whereas COLO 320 does not adhere at all to E-selectin. Finally we provide evidence that monoclonal anti-sialyl-Le(x) can abolish part of the adherence of HL-60 cells to recombinant E-selectin. The adherence of COLO 205 cells can be decreased by either monoclonal anti-sialyl-Le(a) or anti-sialyl-Le(x) antibodies. These results indicate that cell-associated sialylated carbohydrate moieties can act as ligands for recombinant E-selectin.

TNFa‐Induced Expression of Endothelial Adhesion Molecules, ICAM‐1 and VCAM‐1, is Linked to Protein Kinase C Activation

The role of protein kinase C (PKC) in TNFα‐induced activation of endothelial adhesion molecules ICAM‐1 and VCAM‐1 was analysed. Phorbol myristate acetate, which is known to activate PKC, was able lo mimic TNFα‐induced up‐regulation of ICAM‐1 and partly also VCAM‐1 expression. Similarly a PKC inhibitor, H7, but not another kinase inhibitor. HA1004, inhibited TNFα‐induced enhancement of ICAM‐1 expression at both the mRNA and the protein level. Moreover we were able to measure a transient PKC activation peak at 16 min after TNFα induction in endothelial cells analysed by phorbol‐dibutyrate binding. These results indicate that the TNFα‐induced effect on the regulation of endothelial adhesion molecule expression is at least partly mediated by PKC activation.

Trichothecene Mycotoxins Activate Inflammatory Response in Human Macrophages

Damp building-related illnesses have caused concern for years in many countries. Although the problem is extensive, the knowledge of the immunological reactions behind damp building-related illnesses is still quite limited. Trichothecene mycotoxins form one major group of toxins, which possibly contribute to the illnesses. Stachybotrys chartarum is a well-known, but also controversial damp building mold and many strains of this mold are capable of producing trichothecenes. In this report, we have examined the effect of S. chartarum and trichothecene mycotoxins on the proinflammatory cytokine response in human macrophages. As a result, satratoxin-positive S. chartarum activated inflammasome-associated caspase-1, which is needed for proteolytic processing of IL-1β and IL-18. Furthermore, purified trichothecene mycotoxins, roridin A, verrucarin A, and T-2 toxin activated caspase-1, and these mycotoxins also strongly enhanced LPS-dependent secretion of IL-1β and IL-18. The satratoxin-positive strain of S. chartarum and the trichothecenes also triggered the activation of caspase-3, which is an effector caspase of apoptosis. Satratoxin-negative S. chartarum was not able to activate either caspase-1 or caspase-3. In conclusion, our results indicate that human macrophages sense trichothecene mycotoxins as a danger signal, which activates caspase-1, and further enables the secretion of IL-1β and IL-18 from the LPS-primed cells.

Altered MicroRNA Expression of Nasal Mucosa in Long-Term Asthma and Allergic Rhinitis

Background: Asthma and allergic rhinitis (AR) commonly coexist and can be taken as manifestations of one syndrome. Evidence exists that microRNAs (miRNAs) are important in controlling inflammatory processes and they are considered promising biomarkers. However, little is known about the differences in miRNA expression in patients with chronic allergic airway disease. This study evaluated the inflammatory and miRNA profiles of the nasal mucosa of patients with long-term asthma with and without AR. Methods: We analyzed inflammatory cells, cytokines, and miRNAs in nasal biopsies and measured exhaled and nasal nitric oxide levels during the nonpollen season in 117 middle-aged men who had suffered mainly from allergic asthma for approximately 20 years and also in 33 healthy controls. Results: The differences in the number of nasal eosinophils and cytokine expression levels were modest in nasal biopsies taken from asthmatics. Downregulation of miR-18a, miR-126, let-7e, miR-155, and miR-224 and upregulation of miR-498, miR-187, miR-874, miR-143, and miR-886-3p were observed in asthmatic patients in comparison to controls. The differences in miRNA expression were mainly similar in asthmatics with and without AR. With regard to asthma severity, a trend of increased miRNA expression in persistent asthma was seen, whereas the downregulation of certain miRNAs was most distinct in nonpersistent-asthma patients. Conclusions: Differences in miRNA expression in the nasal mucosa of subjects with long-term asthma and AR can be seen also when no markers of Th2-type inflammation are detected. Asthma severity had only a minor impact on miRNA expression.

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLex and sLea respectively) decorated ligands. Endothelial cells have been shown to express sLex epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLex on sialylated N-acetyllactosamine via the action of α(1,3)fucosyltransferase(s), endothelial cells can also degrade sLex to Lewis x through the action of α(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLex, which facilitates their adhesion to endothelial E- and P-selectin.

Decreased cytokine and chemokine mRNA expression in bronchoalveolar lavage in asymptomatic smoking subjects.

Background: Smoking alters the inflammatory cell balance in the airways, often leading to repeated respiratory infections and, eventually, chronic obstructive pulmonary disease (COPD) in susceptible individuals. Objective: It was the aim of this study to evaluate alterations in the airway inflammatory balance caused by chronic cigarette smoke exposure. Methods: We compared results of biopsy and bronchoalveolar lavage (BAL) samples from non-smoking (n = 8) and smoking (n = 5; pack years 25.06 ± 11.75, range 7.13–36.8) subjects without COPD. Results: In BAL samples, we found a significantly higher number of total cells (353 ± 96 million vs. 114 ± 52 million; p = 0.003) and macrophages (331 ± 100 million vs. 84 ± 36 million; p = 0.002) in asymptomatic smoking subjects in comparison with never-smokers. Macrophages correlated negatively with the forced expiratory volume in 1 s as percent of the predicted value (ρ = –0.75, p = 0.003). Of 23 mediators examined, mRNA expression of cytokines interleukin (IL)-6, interferon-γ, tumor necrosis factor-β, IL-13 and chemokines CCL5, CCL3, CCL4 and CCL20 was significantly lowered in BAL cells of smokers compared with never-smokers and was negatively correlated with macrophages and positively correlated with the forced expiratory volume in 1 s as percent of the predicted value. Differential cell counts were similar between smokers and never-smokers in the bronchial biopsies. Conclusion: We conclude that in a susceptible population, smoking suppresses inflammatory defense by inhibiting expression of inflammatory mediators in the airways on a large scale.

Expression of six protein kinase C isotypes in endothelial cells

Protein kinase C (PKC) family is an important regulatory element in signal transduction, cellular regulation and tumor promotion. The classical PKC isotypes (alpha, beta and gamma) are Ca(2+)-dependent and can be activated by diacylglycerol. The novel isotypes, PKC delta, PKC epsilon, PKC eta (L) and PKC theta, are Ca(2+)-independent, whereas the two atypical PKCs (zeta and lambda) lack the Ca(2+)-binding region and are not activated by diacylglycerol. Here we show that cultured human endothelial cell line EA.hy926 as well as freshly isolated human umbilical vein endothelial cells express members of all PKC subfamilies. No traces of PKC gamma or delta were detected in endothelial cells. On the contrary the classical PKCs (alpha and beta), the novel PKC epsilon, as well as the atypical PKC zeta are present at the mRNA level in human endothelial cells and the corresponding proteins are also detected by immunoblotting.

Carcinoembryonic antigen is expressed on endothelial cells

Carcinoembryonic antigen (CEA) is a highly glycosylated cell surface protein. It is produced in large amounts in essentially all colon and several other adenocarcinomas. It has therefore been widely used as a clinical tumor marker. CEA is also a member of the immunoglobulin superfamily. Members of this family, such as ICAM‐1, ICAM‐2, VCAM‐1 and NCAM, are known to participate in cell‐cell adhesion. Similarly, the intercellular adhesion properties of CEA have been documented: it has been shown to mediate homotypic adhesion of cultured human colon adenocarcinoma cell lines. In this study we show for the first time that CEA is expressed on cultured human umbilical vein endothelial cells and on the endothelial cell line Ea.hy926. The expression of CEA on cultured endothelial cells can be enhanced by TNF‐α or IFN‐γ, and decreased by IL‐4. We demonstrate using immunohisto‐chemistry that anti‐CEA monoclonal antibody reacted with FVIII‐positive endothelium in tissue sections prepared from lymph nodes. Finally, we were able to show that CEA‐positive breast carcinoma cells bind to purified CEA protein, whereas CEA‐negative breast carcinoma cells do not. These results reveal for the first time that endothelial cells express CEA on the cell surface and suggest that CEA‐expressing adenocarcinomas could adhere to endothelial cells via CEA‐CEA interaction, thus facilitating tumor cell extravasation and hematogenic metastasis.

Regulation of endothelial adhesion molecules by ligands binding to the scavenger receptor

Monocyte adherence to the endothelium, their penetration to the subendothelial space and excessive lipid accumulation (foam cell formation) are the initial events in atherogenesis. Scavenger receptors have been reported to play an important role in foam cell formation, since modified low density lipoproteins can be taken up via scavenger receptors in a non‐down‐regulated fashion. In this study we demonstrate that stimulation of scavenger receptors in endothelial cells induces the expression of endothelial adhesion molecules. Polyinosinic acid (poly I), a known scavenger receptor ligand, significantly induced the expression of intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1) and E‐selectin on human umbilical vein endothelial cells when compared with polycytidylic acid (poly C), a structurally related compound to poly I, which does not bind to the scavenger receptor. The effect of scavenger receptor ligands on the endothelial cell line EA hy. 926 was also tested. Poly I up‐regutated ICAM‐1 expression also on EA hy. 926 cells, whikit had no effect on IL‐lβ or tumour necrosis factor‐alpha (TNF‐α) production on the same cell line. Poly I‐induced ICAM‐1 expression on EA hy. 926 cells could be inhibited by H7, a protein kinase C inhibitor, while HA 1004, a preferential protein kinase A inhibitor, had no effect on ICAM‐1 expression. The role of protein kinase C in scavenger receptor‐mediated adhesion molecule up‐regulation was confirmed by the ability of poly I to directly activate protein kinase C, when measured with 3H‐phorbol dibutyrate binding to EA hy. 926 cells, while poly C again was ineffective.

Enhanced ICAM-1-Dependent Adhesion of Myelomonocytic Cells Expressing Increased Levels of ß2-Integrins and CD43

Interaction of ICAM‐1 and its ligands plays an important role in the leukocyte binding to endothelium. The best characterized ICAM‐ligands belong to the family of ß2‐integrins (CD11/CD18), but recently it has been suggested that CD43, a molecule with no structural resemblance to integrins binds ICAM‐1 also. On the leukocytes the main regulatory pathway for ICAM‐mediated binding is believed to be a short‐term regulation of the avidity of CD11/CD18. In this study the authors investigated whether a quantitative increase in the surface expression of ICAM‐ligands also can lead to enhanced binding to purified ICAM‐I. PM A‐treatment differentiates myelomonocytic cell lines into macrophages with a concomitant increase in the surface expression and mRNA‐levels of the ß2‐integrin α‐ and ß‐chains as well as that of CD43, another ICAM‐ligand. The binding of the PMA‐treated THP‐1 ceils to ICAM‐1 was increased simultaneously compared to non‐treated cells. The binding was blocked completely with antibodies to CD18 and ICAM‐I. It is concluded that in addition to the transient qualitative regulation, a long‐term quantitative regulation of ICAM‐1 ligands also plays a role in increasing the adhesiveness of myelomonocytic cells. This may be relevant in chronic inflammation episodes.