Adhesion of Lymphoid Cells to CD44-Specific Substrata: The Consequences of Attachment Depend on the Ligand

doi:10.1006/excr.2000.4852

Experimental Cell Research

Volume 256, Issue 2, 1 May 2000, Pages 445-453

https://doi.org/10.1006/excr.2000.4852 Get rights and content

Abstract

Interactions between cell-surface adhesion receptors and immobilized specific substrata can exert profound effects on cell morphology. Using phase-contrast microscopy, we show that CD44-expressing mouse lymphoid cells display a spread morphology when adhering to CD44-specific monoclonal antibody (mAb) immobilized on plastic. This spread morphology is different from that of these same cells when adhering to immobilized hyaluronan, the natural ligand of CD44. Morphometric measurements, in combination with intracellular actin staining and fluorescence microscopy, revealed that the adhesion of lymphoid cells to hyaluronan required essentially no cytoskeletal reorganization and resulted in no fundamental change in morphology. On the other hand, cells adhering to immobilized CD44-specific mAb rearranged their actin structure and established multiple membrane contact sites (spread). Cell spreading on antibody, but not attachment to hyaluronan, was inhibited by cytoskeleton-disrupting agents. Transfection of CD44-negative lymphoid cells with full-length and tailless CD44 enabled these cells to bind to both immobilized hyaluronan and mAb. However, the transfectant lacking the cytoplasmic tail of CD44 spread only transiently on the antibody-coated surface. Our results suggest that CD44 may mediate lymphocyte attachment to its carbohydrate ligand hyaluronan by mechanisms broadly similar to those used by selectins. When immobilized CD44-specific antibody is the ligand, however, CD44 may regulate the activity of the cytoskeleton by mechanisms broadly similar to those used by integrins. In the latter case, the cytoplasmic domain of CD44 contributes to cell spreading.

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Cited by (19)

Interleukin-1β-induced reduction of CD44 Ser-325 phosphorylation in human epidermal keratinocytes promotes CD44 homomeric complexes, binding to ezrin, and extended, monocyte-adhesive hyaluronan coats
2015, Journal of Biological Chemistry
Citation Excerpt :
To explore whether IL-1β induced similar changes in keratinocytes, we performed a scratch wound assay. However, IL-1β did not significantly influence the migratory activity of the keratinocytes (Fig. 6A), suggesting that a robust migration would require additional changes, perhaps enhanced synthesis of hyaluronan (49, 50) or its fixation to substratum (48, 51, 52). Because the hyaluronan coat on keratinocytes was enlarged by IL-1β, we next checked whether this influenced hyaluronan-dependent monocyte binding to the cells.
The proinflammatory cytokine interleukin-1β (IL-1β) attracts leukocytes to sites of inflammation. One of the recruitment mechanisms involves the formation of extended, hyaluronan-rich pericellular coats on local fibroblasts, endothelial cells, and epithelial cells. In the present work, we studied how IL-1β turns on the monocyte adhesion of the hyaluronan coat on human keratinocytes. IL-1β did not influence hyaluronan synthesis or increase the amount of pericellular hyaluronan in these cells. Instead, we found that the increase in the hyaluronan-dependent monocyte binding was associated with the CD44 of the keratinocytes. Although IL-1β caused a small increase in the total amount of CD44, a more marked impact was the decrease of CD44 phosphorylation at serine 325. At the same time, IL-1β increased the association of CD44 with ezrin and complex formation of CD44 with itself. Treatment of keratinocyte cultures with KN93, an inhibitor of calmodulin kinase 2, known to phosphorylate Ser-325 in CD44, caused similar effects as IL-1β (i.e. homomerization of CD44 and its association with ezrin) and resulted in increased monocyte binding to keratinocytes in a hyaluronan-dependent way. Overexpression of wild type CD44 standard form, but not a corresponding CD44 mutant mimicking the Ser-325-phosphorylated form, was able to induce monocyte binding to keratinocytes. In conclusion, treatment of human keratinocytes with IL-1β changes the structure of their hyaluronan coat by influencing the amount, post-translational modification, and cytoskeletal association of CD44, thus enhancing monocyte retention on keratinocytes.
Background: Interleukin-1β recruits leukocytes at the site of inflammation.
Results: The interleukin-1β-induced hyaluronan coat increased monocyte binding to keratinocytes through ezrin-associated CD44 homomers, enabled by reduced serine 325 phosphorylation.
Conclusion: The organization of the cell surface hyaluronan coat is controlled by phosphorylation of CD44.
Significance: Interleukin-1β release in inflamed tissues triggers signals that increase hyaluronan-dependent leukocyte binding.
CD44 interacts directly with Lck in a zinc-dependent manner
2010, Molecular Immunology
CD44 is a widely expressed cell adhesion molecule with functional similarities to the selectin and integrin adhesion molecules. CD44 has a lectin domain that binds hyaluronan, a component of the extracellular matrix. Interactions between CD44 and hyaluronan promote lymphocyte rolling under flow and cell-cell and cell-matrix adhesion. Attachment of lymphocytes to immobilized CD44 antibodies induces cell adhesion and spreading, which is dependent on Src family kinase activity. Both Lck and Fyn associate with CD44 in T cells. CD4 and CD8 associate with Lck via a zinc-dependent interaction that is inhibited by the divalent metal cation chelator, 1,10-phenanthroline. Here we show that both CD4 and CD44-mediated T cell spreading is abolished in the presence of 1,10-phenanthroline and their association with Lck is significantly reduced. In contrast, the co-immunoprecipitation of Fyn by CD44 was unaffected. The cytoplasmic domain of CD44 was required for divalent cation-dependent association of Lck, but not for its association with Fyn. Mutational analysis of CD44 revealed that cysteine residues were not essential for the interaction nor were the carboxy-terminal 41 amino acids. Progressive deletion of the remaining 31 amino acids of the CD44 cytoplasmic domain revealed the importance of this membrane proximal region for its association with Lck. Using purified recombinant proteins, we demonstrated a direct, zinc-inducible interaction between the cytoplasmic domain of CD44 and Lck but not Fyn. The zinc-inducible interaction required the first 13 amino acids of the cytoplasmic domain of CD44 and the non-catalytic regions of Lck. Taken together, we conclude that CD44 directly associates with Lck in a zinc-inducible manner and this is important for the transmission of CD44-mediated signaling events leading to T cell spreading.
Expression of adhesion molecule CD44 on subpopulations of lymphocytes in hypertrophied adenoids in children with otitis media with effusion
2007, Otolaryngologia Polska
The CD44 on lymphocytes binding to its ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on high endothelial venules (HEV). This adhesion molecule plays an important role to maturation and proliferation of lymphocytes in the secondary lymphoid organs. CD44 has a crucial role in migrate of lymphocytes to inflammatory sites. The aim of this study was evaluation of the percentage and MFI (mean fluorescence intensity) of lymphocytes CD4+, CD8+, CD19+ with expression of surface adhesive molecule CD44 in hypertrophied adenoids in children with otitis media with effusion.
42 children with otitis media with effusion and 30 children with hypertrophied adenoids were tested. Children were also divided into two subgroups: young (below 5 years) and older children (above 5 years old). Expression of adhesion molecule CD28 on lymphocytes of adenoids tissue was estimated by flow cytometry method.
This study showed significantly higher percentage of lymphocytes CD19+CD44+ in children with otitis media with effusion (OME 87,83%) than in comparative group with hypertrophied adenoids (AH 85,61%). We did not find difference between OME and AH in mean fluorescence intensity of subpopulations lymphocytes T and B with expression CD44.
Adhesion molecule CD44 is important for developing of effectors lymphocytes in hypertrophy adenoid. Increase percentage of lymphocytes CD19 with expression CD28, especially in older children confirms their participation in developing and forming of immunological response in otitis media with effusion.
Role of the extracellular and cytoplasmic domains of CD44 in the rolling interaction of lymphoid cells with hyaluronan under physiologic flow
2003, Journal of Biological Chemistry
Citation Excerpt :
Both preparations contain high molecular-weight HA (∼1 × 103 and 4 × 103 kDa for umbilical cord HA and Healon®, respectively). Rat monoclonal antibodies (mAb) specific for mouse CD44 IM7.8.1 (referred to as IM7) and IRAWB14 (28-30) were purified from ascites fluid on protein G-Sepharose (Amersham Biosciences, Piscataway, NJ) as described (31, 32). Conjugation of antibodies with biotin (Pierce, Rockford, IL) or Alexa Fluor 594 (Molecular Probes, Eugene, OR) was performed according to the manufacturers' instructions.
CD44 can function as an adhesion receptor that mediates leukocyte rolling on hyaluronan (HA). To study the contributions of different domains of the standard isoform of CD44 to cell rolling, a CD44-negative mouse T lymphoma AKR1 was transfected with wild type (WT) or mutated cDNA constructs. A parallel flow chamber was used to study the rolling behavior of CD44 transfectants on immobilized HA. For CD44WT transfectants, the fraction of cells that rolled and the rolling velocity was inversely proportional to the amount of cell surface CD44. When the cytoplasmic domain distal to Gly³⁰⁵ or sequences that serve as binding sites for cytoskeletal linker proteins, were deleted or replaced with foreign sequences, no significant changes in the rolling behavior of mutant cells, compared with the transfectant expressing CD44WT, were observed. Transfectants lacking 64 amino acids of the cytoplasmic tail distal to Cys²⁹⁵ adhered to HA but showed enhanced rolling at low shear forces. When 83 amino acids from the “non-conserved” membrane-proximal region of the CD44 extracellular domain were deleted, cells adhered firmly to the HA substrate and did not roll at any fluid shear force tested. Unlike wild type cells that exhibited a nearly homogenous distribution of CD44 on a smooth cell surface, cells expressing the non-conserved region deletion mutant accumulated CD44 in membrane protrusions. Disruption of the actin cytoskeleton with cytochalasin B precluded the formation of membrane protrusions, however, treated cells still adhered firmly to HA and did not roll. We conclude that interaction between the cytoplasmic domain of CD44 and the cytoskeleton is not required for cell rolling on immobilized ligand. The strong effect of deletion of the non-conserved region of the extracellular domain argues for a critical role of this region in CD44-dependent rolling and adhesion interactions with HA under flow.
Hyaluronan binding properties of a CD44 chimera containing the link module of TSG-6
2002, Journal of Biological Chemistry
Citation Excerpt :
The AKR1 cell line is a CD44-negative T lymphoma that binds HA constitutively when transfected with wild-type CD44 (59). CTLL-2 is a CD44-negative cytotoxic T cell line that also binds HA constitutively when transfected with CD44 (39). XJ(3)/CD44− is a CD44-negative variant of a pre-B cell line that, when transfected with wild-type CD44, shows an “inducible” HA binding phenotype (37).
CD44, a cell-surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, can mediate leukocyte rolling on hyaluronan substrates and has been implicated in leukocyte migration to sites of inflammation. CD44-mediated binding to hyaluronan is of low affinity, and effective cell/matrix interaction depends on multiple interactions with the multivalent ligand. We replaced the Link module of CD44 with the homologous region of TSG-6, a hyaluronan-binding protein secreted in response to inflammation whose Link module has a higher affinity for ligand. Monoclonal antibodies raised against the CD44/TSG-6 chimera recognized recombinant human TSG-6 and native mouse TSG-6 and blocked hyaluronan binding to these proteins. Cells expressing the CD44/TSG-6 molecule bound hyaluronan with higher avidity than cells expressing CD44. This resulted in changes in the hyaluronan binding properties characteristic of cells expressing CD44 such as requirements for threshold levels of receptor expression and for hyaluronan of high molecular mass. In parallel plate flow assays used to model leukocyte rolling, cells expressing CD44/TSG-6 failed to roll on hyaluronan. Instead, they stuck and remained “tethered” to the substrate under fluid flow. This result argues that the low affinity of CD44 for its ligand is important for rolling, an early phase of leukocyte extravasation from the blood.
Galectin-8 Functions as a Matricellular Modulator of Cell Adhesion
2001, Journal of Biological Chemistry
The interaction of cells with the extracellular matrix regulates cell adhesion and motility. Here we demonstrate that different cell types adhere and spread when cultured in serum-free medium on immobilized galectin-8, a mammalian β-galactoside-binding protein. At maximal doses, galectin-8 is equipotent to fibronectin in promoting cell adhesion and spreading. Cell adhesion to immobilized galectin-8 is mediated by sugar-protein interactions with integrins, and galectin-8 triggers integrin-mediated signaling cascades including Tyr phosphorylation of focal adhesion kinase and paxillin. Cell adhesion is potentiated in the presence of Mn²⁺, whereas it is interrupted in the presence of soluble galectin-8, integrin β₁ inhibitory antibodies, EDTA, or thiodigalactoside but not by RGD peptides. Furthermore, cells readily adhere onto immobilized monoclonal galectin-8 antibodies, which are equipotent to integrin antibodies in promoting cell adhesion. Cell adhesion to immobilized galectin-8 is partially inhibited by serum proteins, suggesting that complex formation between immobilized galectin-8 and serum components generates a matrix that is less supportive of cell adhesion. Accordingly, cell motility on immobilized galectin-8 readily takes place in the presence of serum. Truncation of the C-terminal half of galectin-8, including one of its two carbohydrate recognition domains, largely abolishes its ability to modulate cell adhesion, indicating that both carbohydrate recognition domains are required to maintain a functional form of galectin-8. Collectively, our findings implicate galectin-8 as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of cell surface integrin receptors. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Because of its dual effects on the adhesive properties of the cells and its association with fibronectin, galectin-8 might be considered a novel type of matricellular protein.

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¹: To whom correspondence and reprint requests should be addressed at Department of Orthopedic Surgery, Rush–Presbyterian–St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612. Fax: (312) 942-2101. E-mail: [email protected].

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Regular ArticleAdhesion of Lymphoid Cells to CD44-Specific Substrata: The Consequences of Attachment Depend on the Ligand

Abstract

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Curr. Biol.

Immunology

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Regular Article
Adhesion of Lymphoid Cells to CD44-Specific Substrata: The Consequences of Attachment Depend on the Ligand