Protection of bovine chondrocyte phenotype by heat inactivation of allogeneic serum in monolayer expansion cultures.

Cartilage defects can be addressed with replacement strategies such as autologous chondrocyte implantation (ACI). Expansion of autologous chondrocytes in vitro is an essential step to obtain the necessary cell numbers required for ACI. A major problem with this approach is dedifferentiation of chondrocytes during expansion, resulting in cells with fibroblast-like features. These cells generate cartilage tissue with fibrotic instead of hyaline characteristics. The use of serum is a common feature in most expansion protocols and a potential factor contributing to the dedifferentiation process. The aim of this study was to assess if heat inactivation of serum used in the expansion medium might be a valid approach to generate cells with an improved phenotype and in relevant numbers. We used bovine chondrocyte expansion cultures incubated with heat inactivated allogeneic serum (HIFBS) as a model system. We here show that heat inactivation protects the differentiated phenotype of chondrocytes compared to cultures with regular serum. This is not only true for primary cultures but holds up after two passages. Moreover, using relatively low cell seeding densities, clinically relevant cell numbers can already be reached after the first passage in cultures with HIFBS. In short we here introduce a simple way to improve cell quality while generating relevant amounts of cells during monolayer expansion of bovine chondrocytes in a relative short time period. Our results could have wider implications when translated to the expansion of human chondrocytes.

Culture of primary bovine chondrocytes on a continuously expanding surface inhibits dedifferentiation.

Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and α-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies.

A20 (TNFAIP3) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis.

A20 (TNFAIP3) is a protein that is involved in the negative feedback regulation of NF-κB signaling in response to specific proinflammatory stimuli in different cell types and has been suggested as a susceptibility gene for rheumatoid arthritis. To define the contribution of A20 to rheumatoid arthritis pathology, we generated myeloid-specific A20-deficient mice and show that specific ablation of Tnfaip3 in myeloid cells results in spontaneous development of a severe destructive polyarthritis with many features of rheumatoid arthritis. Myeloid-A20-deficient mice have high levels of inflammatory cytokines in their serum, consistent with a sustained NF-κB activation and higher TNF production by macrophages. Destructive polyarthritis in myeloid A20 knockout mice was TLR4-MyD88 and IL-6 dependent but was TNF independent. Myeloid A20 deficiency also promoted osteoclastogenesis in mice. Together, these observations indicate a critical and cell-specific function for A20 in the etiology of rheumatoid arthritis, supporting the idea of developing A20 modulatory drugs as cell-targeted therapies.

Differential expression of CD97 on human lymphocyte subsets and limited effect of CD97 antibodies on allogeneic T-cell stimulation.

CD97 is a large heptahelical EGF-TM7 receptor broadly expressed on hematopoietic cells as three isoforms with respectively three, four, or five epidermal growth factor (EGF)-like domains. We here describe the expression characteristics of CD97 on human lymphocyte subsets. We found CD97 to be present on all lymphocytes in blood and lymphoid tissue. Expression of CD97 on B cells was lower compared to T and NK cells and did not differ between B-cell subsets. In CD4(+) T cells, CD97 expression was higher on memory cells compared to naive cells. In CD8(+) T and NK cells, we found a downregulation of CD97 on cytolytic effector cells. Stimulation through CD3 and CD28 resulted in a rapid upregulation of CD97 in all T-cell subsets within 2-4h. A link between CD97 expression and lymphocyte proliferation was established in NK cells, which markedly upregulated CD97 in response to IL-2 and IL-15. Mixed lymphocyte cultures revealed a limited ability of the stalk region-specific monoclonal antibody CLB-CD97/3 to inhibit CD8(+) and CD4(+) allogeneic T-cell proliferation.

CD27 defines phenotypically and functionally different human NK cell subsets.

The absence of the TNF-receptor family member CD27 marks the stable acquisition of cytolytic effector functions by both CD4(+) and CD8(+) T cells. We found that the majority of circulating human NK cells was CD27(-). These cells were largely CD56(dim), contained high levels of perforin and granzyme B, and were able to exert strong cytotoxic activity. In contrast, circulating CD27(+) NK cells were mostly CD56(dim/bright), had significant lower levels of perforin and granzyme B, and had a low cytolytic potential. Primary and secondary lymphoid organs were markedly enriched for CD27(+) NK cells. When correlating the expression of CD27 to recently defined developmental stages of NK cells in tonsil, we observed that CD27 was exclusively found on mature CD94(+), stage 4 NK cells. On these cells, regulation of CD27 expression appeared to be controlled by the common gamma-chain cytokine IL-15, and down-regulation of CD27 was specifically induced by its ligand, CD70. Thus, the absence of CD27 expression allows the definition of cytotoxic effector cells within the known mature NK cell subsets in humans.

The human EGF-TM7 receptor EMR3 is a marker for mature granulocytes.

EMR3 is a member of the epidermal growth factor-seven-transmembrane (EGF-TM7) family of adhesion class TM7 receptors. This family also comprises CD97, EMR1, EMR2, and EMR4. To characterize human EMR3 at the protein level, we generated Armenian hamster mAb. Using the mAb 3D7, we here demonstrate that EMR3, like other EGF-TM7 receptors, is expressed at the cell surface as a heterodimeric molecule consisting of a long extracellular alpha-chain, which possesses at its N-terminus EGF-like domains and a membrane-spanning beta-chain. Flow cytometric analysis revealed that all types of myeloid cells express EMR3. In peripheral blood, the highest expression of EMR3 was found on granulocytes. More mature CD16(+) monocytes express high levels of EMR3, and CD16(-) monocytes and myeloid dendritic cells (DC) are EMR3(dim/low). Lymphocytes and plasmacytoid DC are EMR3(-). It is interesting that in contrast with CD97 and EMR2, CD34(+)CD33(-)/CD38(-) committed hematopoietic stem cells and CD34(+)CD33(+)/CD38(+) progenitors in bone marrow do not express EMR3. In vitro differentiation of HL-60 cells and CD34(+) progenitor cells revealed that EMR3 is only up-regulated during late granulopoiesis. These results demonstrate that the expression of EGF-TM7 receptors on myeloid cells is differentially regulated. EMR3 is the first family member found mainly on granulocytes.

An unusual mode of concerted evolution of the EGF-TM7 receptor chimera EMR2.

The epidermal growth factor (EGF)-TM7 receptors CD97, EMR1, EMR2, EMR3, and EMR4 form a group of adhesion class heptahelical molecules predominantly expressed by cells of the immune system. These receptors bind cellular ligands through EGF-like domains, localized N-terminal to a large extracellular region. Remarkably, EMR2 possesses a chimeric structure with a seven-span transmembrane (TM7) region most related to EMR3 and an EGF domain region nearly identical to CD97. By comparing EGF-TM7 receptors in primates and dogs, we identified an intriguing pattern of concerted evolution, apparently mediated by gene conversion, among EMR2 and the oppositely orientated and physically adjacent genes CD97 and EMR3. This concerted evolution has continuously maintained the chimeric structure of EMR2 since early mammal radiation. Most highly conserved between EMR2 and CD97 is the fourth EGF domain, which mediates binding to chondroitin sulfate, a ligand specificity shared by both receptors. Another ligand, CD55, is bound effectively only by CD97. We show that different molecular mechanisms (mutations vs. alternative splicing) prevent CD55 binding by EMR2 in hominoids. Our findings illustrate how various and partially opposing evolutionary events have shaped the structure and ligand specificity of a modern mammalian gene family.

Expression of the largest CD97 and EMR2 isoforms on leukocytes facilitates a specific interaction with chondroitin sulfate on B cells.

The EGF-TM7 receptors CD97 and EMR2 are heptahelical molecules predominantly expressed on leukocytes. A characteristic of these receptors is their ability to interact with cellular ligands via the N-terminal epidermal growth factor (EGF)-like domains. The first two EGF domains of CD97 (but not EMR2) bind CD55 (decay-accelerating factor), while the fourth EGF domain of both CD97 and EMR2 interacts with the glycosaminoglycan chondroitin sulfate (CS). Using fluorescent beads coated with soluble recombinant CD97 and EMR2 protein, and isoform-specific monoclonal antibodies, we have determined the cellular and molecular characteristics of the interaction with CS. The fourth EGF domain of CD97 and EMR2 is expressed on activated lymphocytes and myeloid cells, whereas the ligand is specifically found on B cells within the peripheral blood. The interaction between CD97/EMR2 and CS may therefore play a role in the interaction of activated T cells, dendritic cells, and macrophages with B cells.

The EGF-TM7 family: a postgenomic view.

With the human and mouse genome projects now completed, the receptor repertoire of mammalian cells has finally been elucidated. The EGF-TM7 receptors are a family of class B seven-span transmembrane (TM7) receptors predominantly expressed by cells of the immune system. Within the large TM7 superfamily, the molecular structure and ligand-binding properties of EGF-TM7 receptors are unique. Derived from the processing of a single polypeptide, they are expressed at the cell surface as heterodimers consisting of a large extracellular region associated with a TM7 moiety. Through a variable number of N-terminal epidermal growth factor (EGF)-like domains, EGF-TM7 receptors interact with cellular ligands such as CD55 and chondroitin sulfate. Recent in vivo studies demonstrate a role of the EGF-TM7 receptor CD97 in leukocyte migration. The different number of EGF-TM7 genes in man compared with mice, the chimeric nature of EMR2 and the inactivation of human EMR4 point toward a still-evolving receptor family. Here we discuss the currently available information on this intriguing receptor family.