Elke Wandel

Analysis of CD97 Expression and Manipulation: Antibody Treatment but Not Gene Targeting Curtails Granulocyte Migration

The heptahelical receptor CD97 is a defining member of the EGF-TM7 family of adhesion class receptors. In both humans and mice, CD97 isoforms are expressed with variable numbers of tandemly arranged N-terminal epidermal growth factor-like domains that facilitate interactions with distinct cellular ligands. Results from treatment of mice with mAbs in various disease models have suggested a role for CD97 in leukocyte trafficking. Here, we aimed to thoroughly characterize the expression profile of CD97, and delineate its biological function. To this end, we applied a novel polyclonal Ab, which is the first antiserum suitable for immunohistochemistry, and combined this analysis with the study of Cd97-lacZ knock-in mice. We show that similar to the situation in humans, hematopoietic, epithelial, endothelial, muscle, and fat cells expressed CD97. Despite this broad expression pattern, the Cd97−/− mouse that we created had no overt phenotype, except for a mild granulocytosis. Furthermore, granulocyte accumulation at sites of inflammation was normal in the absence of CD97. Interestingly, application of CD97 mAbs blocked granulocyte trafficking after thioglycollate-induced peritonitis in wild-type but not in knock-out mice. Hence, we conclude that CD97 mAbs actively induce an inhibitory effect that disturbs normal granulocyte trafficking, which is not perturbed by the absence of the molecule.

Thy-1 (CD90) Is an Interacting Partner for CD97 on Activated Endothelial Cells

Leukocyte recruitment in response to inflammatory signals is governed, in part, by binding to Thy-1 (CD90) on activated endothelial cells (EC). In this study, we characterized the adhesion G-protein coupled receptor CD97, present on peripheral myeloid cells, as a novel interacting partner for Thy-1. CD97 was upregulated on polymorphonuclear cells (PMNC) of patients with psoriasis. In psoriatic skin lesions, CD97+ myeloid cells colocalized with Thy-1+ EC of small vessels in microabscesses, suggesting an interaction between CD97 and Thy-1 that was further examined by adhesion and protein-binding assays. PMNC and cell lines stably overexpressing CD97 adhered specifically to Thy-1+–activated human dermal EC, Thy-1+ CHO cells, and immobilized Thy-1 protein. Binding of the CD97+ CHO clones correlated with their CD97 expression level. Soluble CD97 bound specifically to immobilized Thy-1 protein, as well as Thy-1+–activated EC and CHO cells. In all assays, cellular adhesion or protein binding was blocked partially by CD97 and Thy-1–blocking mAb. Our data suggested that CD97 interacts via its stalk with Thy-1 because mAb directed to the stalk of CD97 showed stronger blocking compared with mAb to its epidermal growth factor-like domains, and binding was calcium independent. Moreover, soluble CD97 without the stalk and soluble EMR2, containing highly homologous epidermal growth factor-like domains but a different stalk, failed to bind. In summary, binding of leukocytes to activated endothelium mediated by the interaction of CD97 with Thy-1 is involved in firm adhesion of PMNC during inflammation and may play a role in the regulation of leukocyte trafficking to inflammatory sites.

Glycosaminoglycan-based hydrogels capture inflammatory chemokines and rescue defective wound healing in mice

Glycosaminoglycan-based hydrogels scavenge proinflammatory chemokines, reduce the influx of immune cells, and improve the healing of chronic wounds. Capturing chemokines in chronic wounds Chronic, poor healing wounds are characterized by the lack of resolution of initial proinflammatory signaling present during acute injury. Lohmann et al. designed a synthetic hydrogel wound dressing based on heparin, a glycosaminoglycan that can bind and sequester chemokines. The hydrogel mopped up inflammatory chemokines such as MCP-1 and IL-8 from fluid from patients’ chronic venous leg ulcers in vitro and inhibited neutrophil and monocyte migration. Applying the hydrogel to skin wounds in diabetic mice improved wound healing and vascularization and reduced inflammation more effectively than the FDA-approved hydrogel Promogran. Capturing chemokines may be an effective strategy to promote tissue regeneration in chronic wounds. Excessive production of inflammatory chemokines can cause chronic inflammation and thus impair cutaneous wound healing. Capturing chemokine signals using wound dressing materials may offer powerful new treatment modalities for chronic wounds. Here, a modular hydrogel based on end-functionalized star-shaped polyethylene glycol (starPEG) and derivatives of the glycosaminoglycan (GAG) heparin was customized for maximal chemokine sequestration. The material is shown to effectively scavenge the inflammatory chemokines MCP-1 (monocyte chemoattractant protein–1), IL-8 (interleukin-8), and MIP-1α (macrophage inflammatory protein–1α) and MIP-1β (macrophage inflammatory protein-1β) in wound fluids from patients suffering from chronic venous leg ulcers and to reduce the migratory activity of human monocytes and polymorphonuclear neutrophils. In an in vivo model of delayed wound healing (db/db mice), starPEG-GAG hydrogels outperformed the standard-of-care product Promogran with respect to reduction of inflammation, as well as increased granulation tissue formation, vascularization, and wound closure.

Overexpression of CD97 in intestinal epithelial cells of transgenic mice attenuates colitis by strengthening adherens junctions.

The adhesion G-protein-coupled receptor CD97 is present in normal colonic enterocytes but overexpressed in colorectal carcinoma. To investigate the function of CD97 in colorectal carcinogenesis, transgenic Tg(villin-CD97) mice overexpressing CD97 in enterocytes were generated and subjected to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated tumorigenesis. Unexpectedly, we found a CD97 cDNA copy number-dependent reduction of DSS-induced colitis in Tg compared to wild-type (WT) mice that was confirmed by applying a simple DSS protocol. Ultrastructural analysis revealed that overexpression of CD97 strengthened lateral cell-cell contacts between enterocytes, which, in contrast, were weakened in CD97 knockout (Ko) mice. Transepithelial resistance was not altered in Tg and Ko mice, indicating that tight junctions were not affected. In Tg murine and normal human colonic enterocytes as well as in colorectal cell lines CD97 was localized preferentially in E-cadherin-based adherens junctions. CD97 overexpression upregulated membrane-bound but not cytoplasmic or nuclear β-catenin and reduced phospho-β-catenin, labeled for degradation. This was associated with inactivation of glycogen synthase kinase-3β (GSK-3β) and activation of Akt. In summary, CD97 increases the structural integrity of enterocytic adherens junctions by increasing and stabilizing junctional β-catenin, thereby regulating intestinal epithelial strength and attenuating experimental colitis.

Differential expression of the EGF-TM7 family members CD97 and EMR2 in lipid-laden macrophages in atherosclerosis, multiple sclerosis and Gaucher disease

The members of the epidermal growth factor (EGF)-transmembrane (TM)7 family of adhesion class G-protein coupled receptors are abundantly expressed by cells of the myeloid lineage. A detailed investigation of their expression by functional subsets of activated macrophages is still lacking. Therefore, we determined the expression of CD97, EGF module-containing mucin-like receptor (EMR)2 and EMR3 by monocyte-derived macrophages experimentally polarized in vitro. This was compared to three types of disease-associated lipid-laden macrophages displaying an alternatively activated phenotype in situ. Polarization in vitro towards classically activated M1 versus alternatively activated M2 extremes of macrophage activation did not result in a congruent regulation of EGF-TM7 receptor mRNA and protein except for a down-regulation of CD97 by IL-10. In contrast, macrophages handling lipid overload in vivo displayed differences in the expression of CD97 and EMR2. While foamy macrophages in atherosclerotic vessels expressed both CD97 and EMR2, foam cells in multiple sclerosis brain expressed CD97, but only little EMR2. Foam cell formation in vitro by oxidized LDL and myelin did not affect CD97 or EMR2 expression. Gaucher spleen cells accumulating glucosylceramide expressed very high levels of CD97 and EMR2. These findings indicate that complex cellular expression programmes rather than activation modes regulate the expression of EGF-TM7 receptors in macrophages.

Diversity of CD97 in smooth muscle cells.

CD97, an epidermal growth factor (EGF)-TM7 receptor, is not restricted to hematopoetic and carcinoma cells but is also found on smooth muscle cells (SMC). We have examined its location and biochemical structure in various normal and tumorigenic SMC-containing tissues. SMC of the urinary bladder, lung bronchi and bronchioles, myometrium, and gastrointestinal tract were immunohistologically stained by using monoclonal antibodies (mabs) to the CD97 stalk region (CD97stalk). Mabs directed against an N-glycosylation-dependent epitope within the EGF-domains (CD97EGF) did not bind to normal SMC. Vascular SMC, which was also CD97EGF-negative, showed further CD97 heterogeneity. Only a few, if any, SMC from the aorta or elastic arteries of the systemic circulation were positive for CD97 mRNA and therefore also for CD97stalk. CD97stalk-positive SMC were slightly more numerous in muscular and peripheral arteries. In contrast, most venous SMC expressed CD97stalk. A comparison with other SMC molecules revealed a similar but not identical staining pattern for CD97stalk and desmin. Further CD97 heterogeneity was observed during SMC transformation. All leiomyomas (n=5) and nine out of 21 leiomyosarcomas were positive for both CD97stalk and CD97EGF. As expected, CD97EGF-positive SMC tumors expressed partly N-glycosylated CD97. Seven out of 21 leiomyosarcomas were completely devoid of CD97. Thus, CD97 showed variable expression in vascular and biochemical modification in tumorigenic SMC, suggesting that the function of the molecule is specific for the SMC subtype.

Anti-Inflammatory Action of Keratinocyte-Derived Vaspin: Relevance for the Pathogenesis of Psoriasis

Impaired cross talk between keratinocytes (KCs) and immune cells is believed to contribute to the pathogenesis of chronic inflammatory skin diseases, such as psoriasis. We have previously identified KCs as a rich source of the serpin protease inhibitor vaspin (serpinA12), originally described as an adipokine in adipose tissue. Herein, we studied whether dysregulated vaspin expression in KCs contributes to the pathogenesis of psoriasis. We found vaspin expression to be closely associated to epidermal differentiation, with low levels in proliferating KCs and high levels in differentiated cells. Consistently, in human psoriasis and in a mouse model of a psoriasis-like skin inflammation, epidermal vaspin expression was significantly down-regulated. Down-regulation of vaspin in KCs resulted in decreased expression of differentiation-associated genes and up-regulation of interferon-inducible and inflammation-associated psoriasis signature genes. Vaspin was also shown to modulate the communication between KCs and inflammatory cells under co-culture conditions. A decrease in vaspin expression in KCs stimulated the secretion of tumor necrosis factor-α, IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 by co-cultured dendritic cells, macrophages, monocytes, and neutrophils. Consequently, the application of vaspin inhibited myeloid cell infiltration in a mouse model of a psoriasis-like skin inflammation. In conclusion, vaspin expression by maturing KCs modulates cutaneous immune responses and may be involved in the pathogenesis of psoriasis.

Sulfated hyaluronan attenuates inflammatory signaling pathways in macrophages involving induction of antioxidants

It is well recognized that high molecular weight hyaluronan (H‐HA) exerts potent anti‐inflammatory effects while its fragmentation into low molecular weight HA (L‐HA) is discussed to promote inflammation. Chemical modification of HA with sulfate groups has been shown to foster its anti‐inflammatory activity which seems to be maintained in sulfated low molecular weight HA derivatives (sL‐HA). However, the molecular mechanisms by which sL‐HA produces its anti‐inflammatory activity are not understood. In this study, we used global quantitative proteomics combined with targeted analysis of key proteins to characterize the effect of sL‐HA on fully differentiated human inflammatory macrophages (iMФ). Culture of iMФ with sL‐HA did not affect cell viability but resulted in a reduced pro‐inflammatory cytokine response of iMФ after activation indicating a profound counter‐regulation of their initial inflammatory phenotype. Rapid internalization of sL‐HA involving CD44 and scavenger receptors was observed. Furthermore, an upregulation of the antioxidants SOD2 and SOD3 was found while no oxidative stress was induced. Consequently, activity of transcription factors for inflammatory gene expression was downregulated in iMФ with sL‐HA after activation whereas anti‐inflammatory proteins were induced.

Der Zelloberflächenrezeptor CD97 stimuliert die Migration von Tumorzellen durch Interaktion mit dem Aktin-Zytoskelett

Introduction: The presence of scattered tumor cells at the invasion front of several carcinomas has clinical significance. These cells differ in their protein expression from cells in central tumor regions as shown by us for the EGF- seven transmembrane (TM7) receptor CD97 [1]. We recently clarified the mechanism by which CD97 in scattered tumor cells facilitates tumor invasion and showed that the short CD97 isoform (EGF 1,2,5/TM7) increased random intrinsic migration of tumor cells [2]. Here, we elucidated the molecular mechanisms involved in CD97 stimulated tumor cell migration. Methods: Cells stable overexpressing the whole CD97 (EGF 1,2,5/TM7) molecule, C-terminally truncated CD97 (TM5, TM3, and TM1) as well as CD97 missing the extracellular EGF-domains (delta EGF/TM7) were generated. The cells were applied to migration tracking experiments and laser scanning microscopy to investigate the surface cell distribution of CD97. Moreover, microarray analysis and pulldown-assays were used to define intracellular signaling during migration. Results: In nonconfluent 2D cultured CD97 overexpressing cells, the molecule was concentrated in membrane ruffles at the leading edge of migrating cells, co-localized with the raft marker ganglioside GM1, actin filaments and α-actinin. Treatment that disrupts actin filaments but not microtubules caused loss of CD97 from the ruffles, suggesting an interaction with the actin cytoskeleton in the localization and function of CD97. Eliminating the N-terminal EGF-domains of CD97 diminished migration but did not interrupt it. However, step-wise truncation of CD97 at the C-terminus to TM5 and TM3 decreased random migration. CD97 with only one transmembrane domain (TM1) did not migrate indicating intracellular signaling through the TM7 part during migration. Pull-down assays, microarray analysis and real-time RT-PCR revealed the involvement of the small Rho and Rac GTPases and LIM-kinase 1 in coupling CD97 to actin cytoskeleton formation and thus migration. Conclusion: Malignant cells utilize their intrinsic migratory ability to invade and to metastasize. CD97, upregulated in scattered invasive tumor cells, links migratory signals to the actin cytoskeleton.

The interaction of CD97/ADGRE5 with β-catenin in adherens junctions is lost during colorectal carcinogenesis

The adhesion G-protein-coupled receptor CD97/ADGRE5 is present in adherens junctions of human normal intestinal cells and upregulated in colorectal carcinomas. Here, we examined whether CD97 directly interacts with junctional proteins in normal and malignant colorectal tissue. We identified an association of CD97 with β-catenin using a proximity ligation assay and confirmed the interaction between both endogenous proteins at the biochemical level by co-immunoprecipitation in human and mouse tissues and cell lines. Glutathione S-transferase-pulldown revealed that CD97 binds β-catenin through its seven-span transmembrane/intracellular domain(s). To study tumor-associated changes in the interaction of CD97 and β-catenin in situ, we quantified and correlated both proteins at the membrane, and in the cytoplasm and nuclei of colorectal carcinomas and their corresponding normal tissues (n = 111). In normal colon, membranous levels of CD97 and β-catenin correlated strongly (p < 0.0001). To some degree both molecules disappeared in carcinomas simultaneously from the membrane of tumor cells (p = 0.017). CD97 accumulated in the cytoplasm, whereas β-catenin emerged in the cytoplasm and nuclei. CD97 and β-catenin levels in the cytoplasm correlated well (p < 0.0001). Irrespective of their subcellular localization, interaction of CD97 with β-catenin in tumor cells was also restricted to the cell contacts. Accordingly, CD97 did not regulate β-catenin-dependent TCF-mediated transcriptional activity. In summary, while CD97 and β-catenin interact in adherens junctions, their interaction is lost and both molecules follow different functional paths inside tumor cells.