Anja Saalbach

Human Thy-1 (CD90) on Activated Endothelial Cells Is a Counterreceptor for the Leukocyte Integrin Mac-1 (CD11b/CD18)

Leukocyte recruitment in response to inflammatory signals is in part governed by interactions between endothelial cell receptors belonging to the Ig superfamily and leukocyte integrins. In our previous work, the human Ig superfamily glycoprotein Thy-1 (CD90) was identified as an activation-associated cell adhesion molecule on human dermal microvascular endothelial cells. Furthermore, the interaction of Thy-1 with a corresponding ligand on monocytes and polymorphonuclear cells was shown to be involved in the adhesion of these leukocytes to activated Thy-1-expressing endothelial cells. In this study, we have identified the specific interaction between human Thy-1 and the leukocyte integrin Mac-1 (CD11b/CD18; αMβ2) both in cellular systems and in purified form. Monocytes and polymorphonuclear cells were shown to adhere to transfectants expressing human Thy-1 as well as to primary Thy-1-expressing human dermal microvascular endothelial cells. Furthermore, leukocyte adhesion to activated endothelium as well as the subsequent transendothelial migration was mediated by the interaction between Thy-1 and Mac-1. This additional pathway in leukocyte-endothelium interaction may play an important role in the regulation of leukocyte recruitment to sites of inflammation.

Hyaluronan fragments induce cytokine and metalloprotease upregulation in human melanoma cells in part by signalling via TLR4

Abstract:  Small fragments of the extracellular matrix component hyaluronic acid (sHA) are typically produced at sites of inflammation and tissue injury and have been shown to be associated with tumor invasiveness and metastasis. Here we report that exposure of human melanoma cells to sHA leads to nuclear factor kB (NFk‐B) activation followed by enhanced expression of matrix metalloprotease (MMP) 2 and interleukin (IL)‐8, factors that can contribute to melanoma progression. At the receptor level, we found that Toll‐like receptor (TLR) 4 is involved in this signalling pathway, similar to the case in dendritic and endothelial cells. Specifically, we found that melanoma cells expressed TLR4 on their surface in vivo and in vitro, and using specific siRNA, we could clearly demonstrate the functional importance of TLR4 in sHA‐triggered activation of IL‐8 expression in melanoma cells. Furthermore, we also found that sHA treatment enhanced the motility of melanoma cells, an effect that could again be blocked by TLR4‐specific siRNA. Together, our results suggest that sHA in melanoma might promote tumor invasiveness by inducing MMP‐ and cytokine‐expression, in part in a TLR4‐dependent manner, providing new insights into the relationship between cancer and innate immunity.

The monoclonal antibody AS02 recognizes a protein on human fibroblasts being highly homologous to Thy-1

Abstract Recently, we described a novel fibroblast-restricted monoclonal antibody (mAb AS02) that recognizes a membrane-bound antigen. Characterization and isolation of the corresponding antigen showed that mAb AS02 recognized a protein on human fibroblasts that is highly homologous or identical to human Thy-1 antigen (CD90). Partial amino acid sequencing of the corresponding mAb AS02 antigen and comparison with known proteins revealed a 100% homology of the sequenced peptides to the human Thy-1 antigen. Cross-immunodepletion studies with mAb AS02 and an anti-Thy-1 antibody confirmed these results. Utilizing two-dimensional (2D) gel electrophoresis of fibroblast cell extracts and purified antigen, mAb AS02 and the anti-Thy-1-antibody recognized identical protein spots. Furthermore, we demonstrated many identical biochemical properties of the corresponding AS02 antigen and Thy-1 antigen, such as the molecular weight of the core protein and deglycosylation products and the detection of a GPI anchor. In functional assays, the attachment of fibroblasts to collagen I and fibronectin was increased after incubation of fibroblasts with mAb AS02. Therefore, the Thy-1 antigen appears to be involved in the regulation of the adherence of human dermal fibroblasts.

Interaction of human Thy-1 (CD 90) with the integrin alphavbeta3 (CD51/CD61): an important mechanism mediating melanoma cell adhesion to activated endothelium

The expression of the αvβ3 integrin (CD51/CD61) on human melanoma cells has been shown to be associated most closely with tumor progression and metastases formation in melanoma. Here, we demonstrated a specific interaction of the αvβ3 integrin on melanoma cells with the human Thy-1, an inducible cell adhesion molecule expressed on the cell surface of activated endothelial cells (EC). The interaction was shown by the binding of purified Thy-1 protein to αVβ3 transfected cells, to αvβ3-expressing melanoma cells and to purified αVβ3 integrin. Moreover, melanoma cells adhere specifically to Thy-1 transfectants via αvβ3 on melanoma cells showing the functional relevance of this interaction for cell adhesion. Finally, the importance of the αvβ3/Thy-1 interaction for the adhesion of melanoma cells to the activated endothelium was confirmed under static and flow conditions by the inhibition of melanoma cell adhesion to and transmigration across activated EC by blocking the αvβ3/Thy-1 interaction. In conclusion, we have identified a new pair of adhesion molecules Thy-1 and αvβ3 mediating the interaction of melanoma cells and activated EC. These data explain at least in part the high tumorigenicity of αvβ3-expressing melanoma cells and the association of αvβ3-positive melanoma cells with a high risk of metastasis and poor prognosis.

Detection of human soluble Thy-1 in serum by ELISA. Fibroblasts and activated endothelial cells are a possible source of soluble Thy-1 in serum.

Abstract. The functions of Thy-1, a 35-kDa cell-surface glycoprotein, and its natural ligand are still unknown. Anchoring to the membrane via linkage to phosphatidylinositol (PI) raises the possibility of cleavage off the membrane by PI-specific phospholipases. Soluble Thy-1 (sThy-1) could interfere with the binding of the unknown natural ligand followed by regulation of different cell functions. In this study we established an enzyme-linked immunosorbent assay (ELISA) to measure and quantify sThy-1 in serum and wound fluid. Recombinant human Thy-1 (rhThy-1) was expressed in Drosophila S2 cells, purified from culture supernatant and used as standard for quantitation of sThy-1 by the ELISA technique. There were no differences in sThy-1 levels in serum of healthy donors and patients with systemic sclerosis, leg ulcers, or rheumatoid arthritis, respectively, detected by ELISA. In contrast, at the local site of inflammation, in wound fluid of venous leg ulcers and in synovial fluid from joint puncture, we found strongly elevated levels of sThy-1 compared with sThy-1 in the serum of the same patient. Thy-1 is expressed in humans on brain cells, fibroblasts, a subpopulation of CD34+ blood stem cells, and possibly activated human dermal microvascular endothelial cells. In this study, we never found Thy-1 mRNA or protein expression in resting endothelial cells as shown by reverse transcriptase polymerase chain reaction (RT-PCR) and flow-cytometry. Thy-1 expression could be induced on endothelial cells by phorbol myristate acetate and to a lesser extent by tumor necrosis factor-α (TNF-α). In situ, monoclonal antibodies to Thy-1 did not stain endothelial cells in normal skin, whereas endothelial cells in the synovial membrane of rheumatoid arthritis patients and endothelial cells surrounding melanoma express Thy-1. In summary, our data indicate that Thy-1 is present in soluble form in serum. Furthermore, Thy-1 seems to be a marker for endothelial cell activation. Therefore, activated endothelial cells as well as fibroblasts might be a possible source of sThy-1.

ADAM10 is the constitutive functional sheddase of CD44 in human melanoma cells.

CD44 proteins are cell surface receptors for hyaluronic acid (HA), a component of the extracellular matrix that has multiple effects on cell behavior. CD44 can be shed from the cell surface by proteolytic cleavage. The resulting soluble form can interfere with the interaction between HA and membrane-bound CD44. Soluble CD44 can abolish the cell proliferation-promoting effect of HA on melanoma cell lines, suggesting that a better understanding of the shedding process might identify ways of blocking tumor cell proliferation. ADAM10, ADAM17, and MMP14 have previously been implicated in the shedding of CD44 from various tumor cells. Using immunohistochemistry we demonstrate that ADAM10 and ADAM17 but not MMP14 are significantly expressed on melanoma cells in histological sections. In human melanoma cell lines expression of these proteases could be blocked by transfection with appropriate siRNAs. However, only blocking of ADAM10 expression led to decreased shedding of CD44. In parallel, cell proliferation was promoted. Confocal microscopy demonstrated that ADAM10 and CD44 colocalize on the cell surface. We conclude that ADAM10 is the predominant protease involved in the constitutive shedding of endogenous CD44 from melanoma cells, and that enhancement of ADAM10 activity could be an approach to decrease the proliferation of melanoma cells.

Dermal Fibroblasts Induce Maturation of Dendritic Cells

To trigger an effective T cell-mediated immune response in the skin, cutaneous dendritic cells (DC) migrate into locally draining lymph nodes, where they present Ag to naive T cells. Little is known about the interaction of DC with the various cellular microenvironments they encounter during their migration from the skin to lymphoid tissues. In this study, we show that human DC generated from peripheral blood monocytes specifically interact with human dermal fibroblasts via the interaction of β2 integrins on DC with Thy-1 (CD90) and ICAM-1 on fibroblasts. This induced the phenotypic maturation of DC reflected by expression of CD83, CD86, CD80, and HLA-DR in a TNF-α- and ICAM-1-dependent manner. Moreover, fibroblast-matured DC potently induced T cell activation reflected by CD25 expression and enhanced T cell proliferation. Together these data demonstrate that dermal fibroblasts that DC can encounter during their trafficking from skin to lymph node can act as potent regulators of DC differentiation and function, and thus may actively participate in the regulation and outcome of DC-driven cutaneous immune responses.

Human fibroblasts support the expansion of IL-17–producing T cells via up-regulation of IL-23 production by dendritic cells

The initiation of immune responses is associated with the maturation of dendritic cells (DCs) and their migration to draining lymph nodes. En route activated DCs encounter cells of the tissue microenvironment, such as fibroblasts. Because we have shown that DCs interact with fibroblasts during immune responses, we studied the impact of skin fibroblasts on human monocyte-derived DC function and subsequent human T-cell (TC) differentiation. We show that fibroblasts support interleukin-23 (IL-23) secretion from DCs preactivated by lipopolysaccharide (DC(act)) compared with lipopolysaccharide-activated DCs alone. The underlying complex feedback-loop mechanism involves IL-1β/tumor necrosis factor-α (from DC(act)), which stimulate fibroblasts prostaglandin E(2) production. Prostaglandin E(2), in turn, acts on DC(act) and increases their IL-23 release. Furthermore, fibroblast-stimulated DC(act) are far superior to DC(act) alone, in promoting the expansion of Th17 cells in a Cox-2-, IL-23-dependent manner. Using CD4(+)CD45RO(+) memory TCs and CD4(+)CD45RA(+) naive TCs, we showed that fibroblasts induce a phenotype of DC(act) that promotes the expansion of Th17 cells. Moreover, in psoriasis, a prototypic immune response in which the importance of IL-23/Th17 is known, high expression of Cox-2 in fibroblasts was observed. In conclusion, skin fibroblasts are involved in regulation of IL-23 production in DCs and, as a result, of Th17 expansion.

Norepinephrine-Induced Changes in Cardiac Transforming Growth Factor-β Isoform Expression Pattern of Female and Male Rats

Transforming growth factor-&bgr; (TGF-&bgr;) is a ubiquitous growth-regulating protein with an essential role in tissue repair and formation of extracellular matrix (ECM). To better understand the role of different isoforms of TGF-&bgr; in the cardiac remodeling process induced by norepinephrine (NE), the expression of TGF-&bgr;1, TGF-&bgr;2, and TGF-&bgr;3 was studied and compared with the expression of collagen. NE (0.1 mg/kg · h) was intravenously infused in female and male Sprague-Dawley rats for several time periods, and freshly obtained ventricular myocardium after 1 day was dissociated into myocyte and nonmyocyte fractions. Prazosin (0.1 mg/kg · h) and metoprolol (1 mg/kg · h) were used to block &agr;- and &bgr;-adrenoceptors, respectively. After NE infusion, the three isoforms of TGF-&bgr; were differentially induced as far as the magnitude and the time course is concerned. The increased expression of TGF-&bgr;2 started earlier with a maximum after 12 hours and was more pronounced (10-fold elevation) than that of the other two isoforms, with a clear specificity for the left ventricle in female hearts. This specificity was also seen in male rats with 16-fold elevation of TGF-&bgr;2 after 1 day of NE-stimulation. The increase of TGF-&bgr;2 was significant only in the myocyte fraction obtained from female as well as from male hearts. The expression of the mRNA of all TGF-&bgr; isoforms of collagen type I and type III, and of the matrix metalloproteinase (MMP)-2 and its inhibitor TIMP-2 was reduced predominantly by &agr;-adrenoceptor blockade with prazosin. The increase in TGF-&bgr; isoforms correlated with that of the mRNA expression of collagens, MMP-2 and TIMP-2.

The fibroblast-specific MAb AS02: a novel tool for detection and elimination of human fibroblasts.

Abstract. The unwelcome presence of fibroblasts in many cell cultures prevents the long term cultivation of various cell types and work with pure populations. Recently, we described a novel fibroblast-specific monoclonal antibody (MAb AS02) that recognises a membrane-bound antigen. We have now developed a method using the fibroblast-specific MAb AS02 immobilised on goat-anti-mouse-magnetic beads to separate contaminating fibroblasts. An endothelial cell line experimentally contaminated with 5%–50% fibroblasts was successfully purified. Additionally, an endothelial cell line with an initial fibroblast contamination of 1.5% was prepared. A proportion of each preparation was cultured with no separation step being performed, whereas the remainder was cultured after purification with MAb AS02 to exclude the presence of a minor number of fibroblasts (<0.1%). The proportion of fibroblasts increased up to 38% in the fifth passage of culture without elimination of the low initial fibroblast contamination, whereas in the fraction with the separation step, no fibroblasts were detectable by flow cytometry, even after the fifth passage. We also used the antibody to detect the presence of naturally contaminating fibroblasts in thyrocyte cultures. After cultivation of thyrocyte cultures over five passages, the number of fibroblasts increased dramatically up to 50%–80% of the whole population. Subsequently, we successfully applied the method for complete elimination of naturally contaminating fibroblasts from freshly isolated thyrocyte cultures from enzymatically digested thyroid glands. Thus, MAb AS02 is a fibroblast-specific marker that is a useful tool for the detection and elimination of contaminating fibroblasts. The specificity of MAb AS02 permits the universal application of this antibody for human cell cultures of interest.