Role of sulfation in CD44-mediated hyaluronan binding induced by inflammatory mediators in human CD14(+) peripheral blood monocytes.

Activation of T cells by Ag or stimulation of monocytes with inflammatory cytokines induces CD44 to bind to hyaluronan (HA), an adhesion event implicated in leukocyte-leukocyte, leukocyte-endothelial cell, and leukocyte-stromal cell interactions. We have previously shown that TNF-alpha induces CD44 sulfation in a leukemic cell line, which correlated with the induction of HA binding and CD44-mediated adhesion. In this study, we establish that TNF-alpha and IFN-gamma induce HA binding and the sulfation of CD44 in CD14(+) PBMC, whereas no induced HA binding or CD44 sulfation was observed in CD14(-) PBMC stimulated with TNF-alpha. Treatment of cells with NaClO(3), an inhibitor of sulfation, prevented HA binding in a significant percentage of CD14(+) PBMC induced by TNF-alpha, LPS, IL-1beta, or IFN-gamma. Furthermore, stimulation with TNF-alpha or IFN-gamma in the presence of NaClO(3) reduced the ability of isolated CD44H to bind HA, demonstrating a direct effect of CD44H sulfation on HA binding. In contrast, the transient induction of HA binding in T cells by PHA was not affected by NaClO(3), suggesting that activated T cells do not use sulfation as a mechanism to regulate HA binding. Overall, these results demonstrate that inducible sulfation of CD44H is one mechanism used by CD14(+) peripheral blood monocytes to induce HA binding in response to inflammatory agents such as TNF-alpha and IFN-gamma.     

Oncostatin M and transforming growth factor-beta 1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells

CD44, a receptor for hyaluronan (HA), has been implicated in tumor growth and metastasis. Most CD44-positive cells fail to exhibit constitutive HA receptor function but CD44-mediated HA binding on hematopoetic cells can be induced by antibody cross-linking of the receptor and by physiologic stimuli, including cytokines. We now demonstrate that oncostatin M (OSM) and transforming growth factor-beta1, cytokines known to regulate the growth of tumor cells, stimulate HA binding in lung epithelial-derived tumor cells. In lung epithelial-derived tumor cells, cytokine-induced binding resulted from post-translational modification of the receptor. OSM-induced HA binding was associated with a reduction in N-linked carbohydrate content of CD44. In addition, OSM induced HA binding via a novel mechanism requiring sulfation of chondroitin sulfate chains linked to CD44. The mechanism underlying transforming growth factor-beta1 induced HA binding was distinct from the effects of OSM. The data presented indicate that modulation of the glycosylation and sulfation of CD44 by cytokines provides mechanisms for regulating cell adhesion during tumor growth and metastasis.     

CD44 is a negative regulator of acute pulmonary inflammation and lipopolysaccharide-TLR signaling in mouse macrophages

CD44 is a transmembrane adhesion molecule and hemopoietic CD44 has an essential role in hyaluronan clearance and resolution of noninfectious lung injury. In this study, we examined the role of CD44 in acute pulmonary inflammation and in the regulation of LPS-TLR signaling. Following intratracheally LPS treatment, CD44(-/-) mice demonstrated an exaggerated inflammatory response characterized by increased inflammatory cell recruitment, elevated chemokine expression in bronchoalveolar lavage fluid, and a marked increase in NF-kappaB DNA-binding activity in lung tissue in vivo and in macrophages in vitro. Furthermore, CD44(-/-) mice were more susceptible to LPS-induced shock. Reconstitution of hemopoietic CD44 reversed the inflammatory phenotype. We further found that the induction of the negative regulators of TLR signaling IL-1R-associated kinase-M, Toll-interacting protein, and A20 by intratracheal LPS in vivo and in macrophages in vitro was significantly reduced in CD44(-/-) mice. Collectively, these data suggest CD44 plays a previously unrecognized role in preventing exaggerated inflammatory responses to LPS by promoting the expression of negative regulators of TLR-4 signaling.     

TNFalpha and IL-4 regulation of hyaluronan binding to monocyte CD44 involves posttranslational modification of CD44

Our previous studies have identified TNFalpha as a positive regulator and IL-4 as a negative regulator of human monocyte CD44-HA binding. In order to determine the mechanisms of IL-4- and TNFalpha-mediated regulation of monocyte HA binding, we measured HA binding and CD44 expression on peripheral blood monocytes following monocyte treatment with TNFalpha or IL-4, as well as following monocyte treatment with inhibitors of protein synthesis, N- and O-linked glycosylation, and chondroitin sulfation. IL-4 decreased CD44-HA binding on monocytes initially treated with TNFalpha. Similarly, pretreatment of monocytes with IL-4 prevented subsequent TNFalpha-mediated HA binding. Cycloheximide (protein synthesis inhibitor), tunicamycin (N-linked glycosylation inhibitor), and beta-d-xyloside (chondroitin sulfation inhibitor) all inhibited IL-4-mediated downregulation of TNFalpha-induced monocyte HA binding. Western blot analysis of CD44 from TNFalpha-treated monocytes revealed a 5-10 Mr decrease in the standard isoform of CD44. In contrast, IL-4 treatment of monocytes inhibited CD44-HA binding and reversed the 5- to 10-kDa decrease in monocyte CD44 Mr. Finally, studies with F10.44.2, a CD44 mab that enhances CD44-HA binding, indicated that IL-4 treatment of monocytes not only diminished constitutive HA binding, but also diminished CD44 mab-induced HA binding. Taken together, these data suggested that IL-4-mediated inhibition of TNFalpha-induced monocyte HA binding was dependent not only on protein synthesis, but also on N-linked glycosylation and chondroitin-sulfate modification of either CD44 or, alternatively, another monocyte protein(s) that may regulate the ability of CD44 to bind HA.     

NADPH oxidase-2 derived ROS dictates murine DC cytokine-mediated cell fate decisions during CD4 T helper-cell commitment

NADPH oxidase-2 (Nox2)/gp91(phox) and p47(phox) deficient mice are prone to hyper-inflammatory responses suggesting a paradoxical role for Nox2-derived reactive oxygen species (ROS) as anti-inflammatory mediators. The molecular basis for this mode of control remains unclear. Here we demonstrate that IFNγ/LPS matured p47(phox-/-)-ROS deficient mouse dendritic cells (DC) secrete more IL-12p70 than similarly treated wild type DC, and in an in vitro co-culture model IFNγ/LPS matured p47(phox-/-) DC bias more ovalbumin-specific CD4(+) T lymphocytes toward a Th1 phenotype than wild type (WT) DC through a ROS-dependent mechanism linking IL-12p70 expression to regulation of p38-MAPK activation. The Nox2-dependent ROS production in DC negatively regulates proinflammatory IL-12 expression in DC by constraining p38-MAPK activity. Increasing endogenous H(2)O(2) attenuates p38-MAPK activity in IFNγ/LPS stimulated WT and p47(phox-/-) DC, which suggests that endogenous Nox 2-derived ROS functions as a secondary messenger in the activated p38-MAPK signaling pathway during IL-12 expression. These findings indicate that ROS, generated endogenously by innate and adaptive immune cells, can function as important secondary messengers that can regulate cytokine production and immune cell cross-talk to control during the inflammatory response.     

TNF-alpha increases the carbohydrate sulfation of CD44: induction of 6-sulfo N-acetyl lactosamine on N- and O-linked glycans

CD44 and sulfation have both been implicated in leukocyte adhesion. In monocytes, the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) stimulates CD44 sulfation, and this correlates with the induction of CD44-mediated adhesion events. However, little is known about the sulfation of CD44 or its induction by inflammatory cytokines. We determined that TNF-alpha induces the carbohydrate sulfation of CD44. CD44 was established as a major sulfated cell surface protein on myeloid cells. In the SR91 myeloid cell line, the majority of CD44 sulfation was attributed to the glycosaminoglycan chondroitin sulfate. However, TNF-alpha stimulation increased CD44 sulfation two- to threefold, largely attributed to the increased sulfation of N- and O-linked glycans on CD44. Therefore, TNF-alpha induced a decrease in the percentage of CD44 sulfation due to chondroitin sulfate and an increase due to N- and O-linked sulfation. Furthermore, TNF-alpha induced the expression of 6-sulfo N-acetyl lactosamine (LacNAc)/Lewis x on these cells, which was detected by a monoclonal antibody after neuraminidase treatment. This 6-sulfo LacNAc/Lewis x epitope was induced on N-linked and (to a lesser extent) on O-linked glycans present on CD44. This demonstrates that CD44 is modified by sulfated carbohydrates in myeloid cells and that TNF-alpha modifies both the type and amount of carbohydrate sulfation occurring on CD44. In addition, it demonstrates that TNF-alpha can induce the expression of 6-sulfo N-acetyl glucosamine on both N- and O-linked glycans of CD44 in myeloid cells.     

Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding

CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding.     

Differing signal requirements for the activation of macrophages from C3H/HeJ and C3H/OuJ mice

Abstract Endotoxin-associated protein (EP) from Salmonella typhi stimulated the release of prostaglandin E2 (PGE2), interleukin-1 (IL-1), and interferon (IFN) activity in macrophages from the lipopolysaccharide (LPS) responder C3H/OuJ mouse strain. However, only PGE2 and IL-1 were stimulated by EP in macrophages from the LPS nonresponder C3H/HeJ mouse strain. LPS stimulated the release of PGE2, IL-1 and IFN activity in C3H/OuJ macrophages, but not in C3H/HeJ macrophages. The protein kinase C (PKC) activator phorbol myristic acid (PMA) stimulated PGE2 production in both strains but not IL-1 production, suggesting that signalling pathways other than PKC may be involved in IL-1 production. The calcium ionophore ionomycin stimulated PGE2 production in C3H/OuJ but not C3H/HeJ macrophages, suggesting a defective calcium-related pathway in the C3H/HeJ macrophages as compared to the C3H/OuJ cells.     

Regulation of proximal tubular epithelial cell CD44-mediated binding and internalisation of hyaluronan

BACKGROUND: Increased expression of the connective tissue polysaccharide hyaluronan (HA) in the renal corticointerstitium is associated with progressive renal fibrosis. Numerous studies have demonstrated involvement proximal tubular epithelial cells in the fibrotic process and in the current study we have characterised their expression of the HA receptor, CD44, and examined changes in CD44 expression and function in response to either IL-1beta or glucose. METHODS: Characterisation of CD44 splice variant expression was carried out in primary cultures of human proximal tubular cells (PTC) and HK2 cells. Binding and internalisation HA was examined by addition of exogenous of fluorescein-HA (fl-HA), and expression of CD44 examined by immunoblot analysis and flow cytometry. Alteration in "functional" CD44 was determined by immunoprecipitation of CD44 following stimulation in the presence of fl-HA. RESULTS: PTC, both primary culture and the PTC cell line, HK2, express at least 5 CD44 splice variants, the expression of which are not altered by addition of either IL-1beta or 25mM D-glucose. Addition of either stimulus increased cell surface binding and internalisation of fl-HA and increased expression of functionally active CD44. Increased binding and internalisation of fl-HA, was blocked by anti-CD44 antibody, and by the inhibition of O-glycosylation. CONCLUSIONS: The data demonstrate that stimuli inducing PTC HA synthesis also regulate PTC-HA interactions. Furthermore increased HA binding and internalisation is the result of post-translational modification of CD44 by O-glycosylation, rather than by alteration in expression of CD44 at the cell surface, or by alternate use of CD44 splice variants.     

Adiponectin induces TNF-alpha and IL-6 in macrophages and promotes tolerance to itself and other pro-inflammatory stimuli

Adiponectin exerts anti-inflammatory effects via macrophages, suppressing the production of pro-inflammatory cytokines in response to bacterial lipopolysaccharide (LPS). Here, we provide experimental evidence that the "anti-inflammatory" effect of adiponectin may be due to an induction of macrophage tolerance: globular adiponectin (gAd) is a powerful inducer of TNF-alpha and IL-6 secretion in primary human peripheral macrophages, in the THP-1 human macrophage cell line, and in primary mouse peritoneal macrophages. Pre-exposure of macrophages to 10 microg/ml gAd rendered them tolerant to further gAd exposure or to other pro-inflammatory stimuli such as TLR3 ligand polyI:C and TLR4 ligand LPS, while pre-exposure to 1 microg/ml of and re-exposure to 10 microg/ml gAd unmasked its pro-inflammatory properties. GAd induced NF-kappaB activation and tolerance to further gAd or LPS exposure. Our data suggest that adiponectin constant presence in the circulation in high levels (in lean subjects) renders macrophages resistant to pro-inflammatory stimuli, including its own.     

Hyaluronan in part mediates IL-1beta-induced inflammation in mouse chondrocytes by up-regulating CD44 receptors

Interleukin-1beta (IL-1beta) elicits the expression of inflammatory mediators through a mechanism involving the CD44 receptor. Hyaluronan (HA) depolymerization also contributes to CD44 activation. This study investigated the potential of HA fragments, obtained by hyaluronidase (HYAL) treatment, as mediators of CD44 activation on IL-1beta-induced inflammation in mouse chondrocytes.mRNA and related protein levels were measured for CD44, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13) and inducible nitric oxide synthase (iNOS) in chondrocytes, treated or untreated with IL-1beta, either with or without the addition of HYAL. The level of NF-kB activation was also assayed.CD44 mRNA expression was higher than controls in chondrocytes treated with IL-1beta. IL-1beta also induced NF-kB up-regulation and increased TNF-alpha, IL-6, MMP-13 and iNOS expression. Different effects resulted from HYAL treatment. Treatment of chondrocytes exposed to IL-1beta with HYAL synergistically increased the same parameters up-regulated by IL-1beta, while the same parameters were increased by HYAL in chondrocytes not exposed to IL-1beta but to a lesser extent. Specific CD44 blocking antibody and hyaluronan binding protein (HABP), which inhibit HA activity, were used to confirm CD44 to be the target of IL-1beta action through HA mediation. HA levels and molecular size further confirm the role of degraded HA.These findings suggest that IL-1beta exerts inflammatory activity via CD44 by the mediation of HA fragments derived from HA depolymerization.     

Distinct Kinetic and Molecular Requirements Govern CD44 Binding to Hyaluronan versus Fibrin(ogen)

CD44 is a multifunctional glycoprotein that binds to hyaluronan and fibrin(ogen). Alternative splicing is responsible for the generation of numerous different isoforms, the smallest of which is CD44s. Insertion of variant exons into the extracellular membrane proximal region generates the variant isoforms (CD44v). Here, we used force spectroscopy to delineate the biophysical and molecular requirements of CD44-HA and CD44-fibrin(ogen) interactions at the single-molecule level. CD44v-HA and CD44s-HA single bonds exhibit similar kinetic and micromechanical properties because the HA-binding motif on CD44 is common to all of the isoforms. Although this is the primary binding site, O- and N-linked glycans and sulfation also contribute to the tensile strength of the CD44-HA bond. The CD44s-fibrin pair has a lower unstressed dissociation rate and a higher tensile strength than CD44s-fibrinogen but is weaker than the CD44-HA bond. In contrast to CD44-HA binding, the molecular interaction between CD44 and fibrin(ogen) is predominantly mediated by the chondroitin sulfate and dermatan sulfate on CD44. Blocking sulfation on CD44s modestly decreases the tensile strength of CD44s-fibrin(ogen) binding, which is in stark contrast to CD44v-fibrin interaction. Collectively, the results obtained by force spectroscopy in conjunction with biochemical interventions enable us to delineate the biophysical parameters and molecular constituents of CD44 binding to hyaluronan and fibrin(ogen).     

Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells

Proinflammatory cytokines such as TNF-alpha up-regulate the expression of the cell adhesion molecule, CD44, and induce hyaluronan (HA) binding in peripheral blood monocytes (PBM). Here we show that in PBM, TNF-alpha induced cytoskeletal rearrangement, increased threonine phosphorylation of ERM proteins, and induced the redistribution and colocalization of phospho-ERM proteins (P-ERM) with CD44. In the myeloid progenitor cell line, KG1a, hyaluronan binding occurred in the pseudopod where CD44, P-ERM, and F-actin were highly localized. Hyaluronan binding correlated with high expression of both CD44 and P-ERM clustered in a single pseudopod. Disruption of polymerized actin reduced hyaluronan binding in both PBM and KG1a cells and abolished CD44 clustering and the pseudopod in KG1a cells. The pseudopod was not required for the clustering of CD44, the colocalization with P-ERM, or hyaluronan binding. However, treatment with a kinase inhibitor abolished ERM phosphorylation and reduced hyaluronan binding. Furthermore, expression of CD44 lacking the putative ERM binding site resulted in reduced hyaluronan binding. Taken together, these data suggest that CD44-mediated hyaluronan binding in human myeloid cells is regulated by P-ERM and the actin cytoskeleton.     

CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxol-inducible gene expression

Overproduction of inflammatory mediators by macrophages in response to Gram-negative LPS has been implicated in septic shock. Recent reports indicate that three membrane-associated proteins, CD14, CD11b/CD18, and Toll-like receptor (TLR) 4, may serve as LPS recognition and/or signaling receptors in murine macrophages. Therefore, the relative contribution of these proteins in the induction of cyclooxygenase 2 (COX-2), IL-12 p35, IL-12 p40, TNF-alpha, IFN-inducible protein (IP)-10, and IFN consensus sequence binding protein (ICSBP) genes in response to LPS or the LPS-mimetic, Taxol, was examined using macrophages derived from mice deficient for these membrane-associated proteins. The panel of genes selected reflects diverse macrophage effector functions that contribute to the pathogenesis of septic shock. Induction of the entire panel of genes in response to low concentrations of LPS or Taxol requires the participation of both CD14 and TLR4, whereas high concentrations of LPS or Taxol elicit the expression of a subset of LPS-inducible genes in the absence of CD14. In contrast, for optimal induction of COX-2, IL-12 p35, and IL-12 p40 genes by low concentrations of LPS or by all concentrations of Taxol, CD11b/CD18 was also required. Mitigated induction of COX-2, IL-12 p35, and IL-12 p40 gene expression by CD11b/CD18-deficient macrophages correlated with a marked inhibition of NF-kappa B nuclear translocation and mitogen-activated protein kinase (MAPK) activation in response to Taxol and of NF-kappa B nuclear translocation in response to LPS. These findings suggest that for expression of a full repertoire of LPS-/Taxol-inducible genes, CD14, TLR4, and CD11b/CD18 must be coordinately engaged to deliver optimal signaling to the macrophage.     

Interferon gamma induces actin polymerization, Rac1 activation and down regulates phagocytosis in human monocytic cells

IFNγ is a potent activator and IL-10 a powerful inhibitor of macrophage functions. However, neither all cellular functions are enhanced by IFNγ nor IL-10 inhibits all cellular responses. Thus, FcγRs-mediated phagocytosis in monocyte-derived macrophages (MDM) increases after IL-10 treatment, and decreases after treatment with IFNγ, although both IL-10 and IFNγ up regulate FcγRI expression. In this work we investigated the effect of IFNγ and IL-10 on phagocytic signaling by FcγRs in MDM. Treatment with IFNγ diminished phagocytosis of IgG-opsonized SRBC (IgG-SRBC) while treatment with IL-10 increased it. These opposite effects cannot be attributed to changes in FcγR expression induced by each cytokine. Early biochemical responses mediated by FcγRs were distinctly affected by cytokine treatment. Syk phosphorylation and the rise in [Ca²⁺]_i were higher after IL-10 treatment, whereas IFNγ treatment also increased Syk phosphorylation but had no effect on the rise in [Ca²⁺]_i. IFNγ treatment led to increased basal levels of F-actin and this effect correlated with the decrease in phagocytosis of both IgG-SRBC and non-opsonized Escherichia coli. IL-10 did not alter F-actin basal levels, and enhanced the phagocytosis of E. coli and IgG-SRBC. The level of F-actin reached after IFNγ treatment was not further increased after stimulation with IgG-SRBC or CCL5, whereas MDM treated with IL-10 showed a slightly higher response than control cells to CCL5. IFNγ increased Rac1-GTP levels. Inhibition of PI3K with LY294002 prevented IFNγ-mediated actin polymerization. Our data suggest that IFNγ induces a higher basal level of F-actin and activation of Rac1, affecting the response to stimuli that induce cytoskeleton rearrangement such as phagocytic or chemotactic stimuli.     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.