Deficiency of hyaluronan synthase 1 (Has1) results in chronic joint inflammation and widespread intra-articular fibrosis in a murine model of knee joint cartilage damage.

OBJECTIVE: Articular cartilage defects commonly result from traumatic injury and predispose to degenerative joint diseases. To test the hypothesis that aberrant healing responses and chronic inflammation lead to osteoarthritis (OA), we examined spatiotemporal changes in joint tissues after cartilage injury in murine knees. Since intra-articular injection of hyaluronan (HA) can attenuate injury-induced osteoarthritis in wild-type (WT) mice, we investigated a role for HA in the response to cartilage injury in mice lacking HA synthase 1 (Has1(-/-)). DESIGN: Femoral groove cartilage of WT and Has1(-/-) mice was debrided to generate a non-bleeding wound. Macroscopic imaging, histology, and gene expression were used to evaluate naïve, sham-operated, and injured joints. RESULTS: Acute responses (1-2 weeks) in injured joints from WT mice included synovial hyperplasia with HA deposition and joint-wide increases in expression of genes associated with inflammation, fibrosis, and extracellular matrix (ECM) production. By 4 weeks, some resurfacing of damaged cartilage occurred, and early cell responses were normalized. Cartilage damage in Has1(-/-) mice also induced early responses; however, at 4 weeks, inflammation and fibrosis genes remained elevated with widespread cartilage degeneration and fibrotic scarring in the synovium and joint capsule. CONCLUSIONS: We conclude that the ineffective repair of injured cartilage in Has1(-/-) joints can be at least partly explained by the markedly enhanced expression of particular genes in pathways linked to ECM turnover, IL-17/IL-6 cytokine signaling, and apoptosis. Notably, Has1 ablation does not alter gross HA content in the ECM, suggesting that HAS1 has a unique function in the metabolism of inflammatory HA matrices.

Acute and chronic rejection: compartmentalization and kinetics of counterbalancing signals in cardiac transplants.

Acute and chronic rejection impact distinct compartments of cardiac allografts. Intramyocardial mononuclear cell infiltrates define acute rejection, whereas chronic rejection affects large arteries. Hearts transplanted from male to female C57BL/6 mice undergo acute rejection with interstitial infiltrates at 2 weeks that resolve by 6 weeks when large arteries develop arteriopathy. These processes are dependent on T cells because no infiltrates developed in T cell-deficient mice and transfer of CD4 T cells restored T cell as well as macrophage infiltrates and ultimately neointima formation. Markers of inflammatory macrophages were up-regulated in the interstitium acutely and decreased as markers of wound healing macrophages increased chronically. Programmed cell death protein, a negative costimulator, and its ligand PDL1 were up-regulated in the interstitium during resolution of acute rejection. Blocking PDL1:PD1 interactions in the acute phase increased interstitial T cell infiltrates. Toll-like receptor (TLR) 4 and its endogenous ligand hyaluronan were increased in arteries with neointimal expansion. Injection of hyaluronan fragments increased intragraft production of chemokines. Our data indicate that negative costimulatory pathways are critical for the resolution of acute interstitial infiltrates. In the arterial compartment recognition of endogenous ligands including hyaluronan by the innate TLRs may support the progression of arteriopathy.

Characterization of hyaluronan cable structure and function in renal proximal tubular epithelial cells.

Alteration in the glycosaminoglycan hyaluronan (HA) has been demonstrated in numerous renal diseases. We have demonstrated that renal proximal tubular epithelial cells (PTCs) surround themselves in vitro with HA in an organized pericellular matrix or 'coat', which is associated with cell migration, and also form pericellular HA cable-like structures which modulate PTC-mononuclear leukocytes interactions. The aim of this study was to characterize potential regulatory mechanism in the assembly of PTC-HA into pericellular cables. HA cables are generated by PTCs in the absence of serum. Immunohistochemical analysis demonstrates the incorporation of components of the inter-alpha-inhibitor (IalphaI) family of proteins and versican into HA cables. Addition of an antibody to IalphaI/PalphaI (pre-alpha-inhibitor) inhibits cable formation. In contrast, inhibition of tumor necrosis factor-alpha-stimulated gene 6 (TSG-6) has no effect on cable formation, suggesting that their generation is independent of the known heavy-chain transfer activity of TSG-6. Overexpression of HAS3 is associated with induction of HA cable formation, and also increased incorporation of HA into pericellular coats. Functionally, this resulted in enhanced HA-dependent monocyte binding and cell migration, respectively. Cell surface expression of CD44 and trypsin-released cell-associated HA were increased in HAS3-overexpressing cells. In addition, hyaluronidase (hyal1 and hyal2) and bikunin mRNA expression were increased, whereas PalphaI HC3 mRNA expression was unchanged in the transfected cells. The data demonstrate the importance of IalphaI/PalphaI in cable formation and suggest that expression of HAS3 may be critical for HA cable assembly.

Induction of E-selectin expression by double-stranded RNA and TNF-alpha is attenuated in murine aortic endothelial cells derived from double-stranded RNA-activated kinase (PKR)-null mice.

The adherence of leukocytes on the endothelium is mediated in part by the transient expression of the E-selectin adhesion molecule. Because we have previously shown that the dsRNA-activated kinase PKR mediates dsRNA induction of NF-kappaB, we used murine aortic endothelial (MuAE) cells isolated from wild-type and PKR-null mice to investigate the role of PKR in the induction of E-selectin expression by dsRNA (pIC) and TNF-alpha. E-selectin mRNA and protein expression was inducible by both pIC and TNF-alpha in wild-type MuAE cells, whereas induction of E-selectin expression by these agents was defective in PKR-null MuAE cells. Induction of E-selectin promoter activity and NF-kappaB DNA binding activity were substantially reduced in pIC- or TNF-alpha-treated PKR-null cells, indicating a role for PKR in both pIC and TNF-alpha induction of E-selectin via an NF-kappaB-dependent pathway. In PKR-null cells, pIC-mediated degradation of IkappaBbeta is deficient. Activation of this pathway requires the PKR-dependent degradation of the IkappaBbeta protein. Moreover, both phosphorylated and unphosphorylated activating transcription factor 2 DNA-binding activities were reduced in PKR-null aortic endothelial cells. These results indicate that the PKR is required for full activation of E-selectin expression by pIC and TNF-alpha in primary mouse aortic endothelial cells identifying activating transcription factor 2 as a new target for PKR-dependent regulation and suggest a role for PKR in leukocyte adhesion.

Mononuclear leukocytes preferentially bind via CD44 to hyaluronan on human intestinal mucosal smooth muscle cells after virus infection or treatment with poly(I.C).

Pathological changes in inflammatory bowel disease include an increase in intestinal mucosal mononuclear leukocytes and hyperplasia of the muscularis mucosae smooth muscle cells (M-SMCs). Because virus infections have correlated with disease flare, we tested the response of cultured M-SMCs to respiratory syncytial virus, measles virus, and the viral analogue, poly(I.C). Adhesion of U937 cells and peripheral blood mononuclear cells was used to measure the leukocyte-interactive potential of M-SMCs. Untreated M-SMCs, only minimally adhesive for leukocytes, bound U937 cells after treatment with respiratory syncytial virus or measles virus. Mononuclear leukocytes also bound to poly(I.C)-treated M-SMCs. Although both vascular cell adhesion molecule-1 mRNA and protein increased 3-4-fold in poly(I.C)-treated M-SMC cultures, U937 cell adhesion was not blocked by an anti-vascular cell adhesion molecule-1 monoclonal antibody. However, hyaluronidase digestion of poly(I.C)- or virus-treated M-SMCs dramatically reduced leukocyte adhesion ( approximately 75%). Fluorophore-assisted carbohydrate electrophoresis demonstrated a approximately 3-fold increase in surface-bound hyaluronan on poly(I.C)-treated M-SMCs compared with untreated controls. In addition, pretreatment of mononuclear cells with a blocking anti-CD44 antibody, greatly decreased adhesion to poly(I.C)-treated M-SMCs. Recognition of this virus-induced hyaluronan/CD44 mechanism of mesenchymal cell/leukocyte interaction introduces a new avenue in the research of gut inflammation.

Differential monocytic cell adherence to specific anatomic regions of the canine aorta.

Circulating monocytes and vascular endothelial cells (EC) interact in a complex and dynamic manner that varies between vascular beds. The objective of this study was twofold: to ascertain if monocytic cell adhesion to vascular endothelium differed between specific anatomic regions of the canine aorta, and to investigate the effect of known EC stimulators on monocytic cell adhesion to cells from these regions. Initial in vitro studies measuring adherence of U937 cells, a human monocytic cell line, to canine jugular vein and aortic EC monolayers revealed a dose-dependent increase in adhesion to EC stimulated with interleukin-1 (IL-1), lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), or thrombin. While there was no regional difference in monocytic cell adherence to unstimulated EC in tissue culture, studies demonstrated greater monocytic cell adhesion to stimulated EC cultured from the distal versus proximal aorta. In organ culture, unstimulated adhesion of U937 cells or autologous monocytes was significantly greater to the distal aorta than the proximal aorta. Although monocytic cell adhesion to both the proximal and distal aorta increased with stimulation, the percentage increase in the proximal aorta, 1,086% with IL-1, 237% with PMA, 209% with LPS, and 174% with thrombin, was greater than in the distal aorta, demonstrating a significant functional difference in the endothelium from separate anatomic regions of a single vessel. This may have a direct relevance to the regional specificity of vascular disease.

Thrombin receptor-activating peptides differentially stimulate platelet-derived growth factor production, monocytic cell adhesion, and E-selectin expression in human umbilical vein endothelial cells.

Recent studies have shown that the synthetic peptides SFL LRN and SFL LRN PND KYEPF (thrombin receptor-activating peptides (TRAP)) derived from the deduced sequence of the new amino terminus of the cleaved thrombin receptor can mimic thrombin receptor activation, act as full agonists for platelet activation, and induce prostaglandin I2 production as well as cytosolic Ca2+ increase in human umbilical vein endothelial cells (HUVEC). Here, we have compared the ability of these synthetic peptide ligands and thrombin to stimulate platelet-derived growth factor (PDGF) production by, and monocyte adhesion to, HUVEC. Thrombin (50 units/ml) and TRAP (25 microM) maximally stimulated monocyte adhesion. Furthermore, the stimulation of E-selectin cell surface expression and the steady-state E-selectin mRNA levels by thrombin and TRAP were comparable. Thrombin (50 units/ml) stimulated PDGF production 400% above the basal level in 24 h, whereas the 6-mer and 14-mer TRAP, even at 200 microM, did not significantly stimulate PDGF production. Northern analysis, however, revealed that TRAP at 100 microM stimulated PDGF-A and -B chain mRNA expression to a level similar to that induced by thrombin. These results suggest that activation of cell signaling by TRAP can mimic thrombin and is sufficient for the stimulation of monocyte adhesion to HUVEC; however, thrombin-stimulated PDGF production by HUVEC may require mechanisms in addition to the signaling events initiated by TRAP or may require the participation of a novel thrombin receptor.

3-Deazaadenosine inhibits thrombin-stimulated platelet-derived growth factor production and endothelial-leukocyte adhesion molecule-1-mediated monocytic cell adhesion in human aortic endothelial cells.

Injury to the vascular endothelium and the subsequent inflammatory response are considered prerequisites for the development of atherosclerosis. Platelet-derived growth factor (PDGF) production by and monocyte adhesion to aortic endothelial cells (EC) may participate in this inflammatory process and therefore are two potential targets for control by anti-inflammatory agents. Our previous studies have demonstrated that monocyte adhesion and PDGF production are stimulated by thrombin in EC. Here, we provide evidence that treatment of EC with the anti-inflammatory agent 3-deazaadenosine (c3Ado) effectively abolished thrombin-stimulated PDGF production and monocyte adhesion. c3Ado had no significant effect on either basal monocyte adhesion or constitutive PDGF production. c3Ado was also effective in negating monocyte adhesion induced by other agonists, such as interleukin-1, phorbol 12-myristate 13-acetate (PMA), and lipopolysaccharide. Northern analysis demonstrated that c3Ado significantly reduced thrombin- and PMA-stimulated steady-state levels of PDGF-A chain, PDGF-B chain, and endothelial-leukocyte adhesion molecule-1 (ELAM-1) mRNAs. Nuclear run-on studies demonstrated that a marked transcriptional activation of these genes by thrombin and PMA was abrogated by c3Ado treatment. The transcriptional rate of the alpha-tubulin gene was unaffected by the drug. Antibody binding studies with an anti-ELAM-1 monoclonal antibody 7A9 revealed that thrombin-stimulated EC expression of ELAM-1 was abolished by c3Ado, indicating that the suppression of ELAM-1 expression on EC surface may be a mechanism by which c3Ado interferes with monocyte adhesion. Experiments with the nucleoside transport inhibitor nitrobenzylthioinosine suggested that the transport of c3Ado into EC was required for its inhibitory activity. In addition, L-homocysteine thiolactone was found to potentiate the inhibitory activity of c3Ado, suggesting that the accumulation of intracellular c3Ado homocysteine may be the underlying mechanism by which c3Ado inhibits thrombin-induced EC function. Taken together, these results indicate that c3Ado may prove effective against vascular injury and inflammation through its ability to inhibit induction of both monocyte adhesion and PDGF production.

Thrombin stimulates PDGF production and monocyte adhesion through distinct intracellular pathways in human endothelial cells.

Thrombin stimulates multiple functions in cultured endothelial cells (EC), including an increase in cell surface adhesion sites for monocytes and the production of platelet-derived growth factor (PDGF). We have initiated studies to define the intracellular signaling pathways involved in these two thrombin-induced EC functions by focusing on the possible roles of the Na(+)-H+ antiporter and guanine nucleotide-binding proteins (G proteins). Amiloride suppressed thrombin-stimulated PDGF production by human aortic EC without affecting either basal PDGF production or overall protein synthesis. The steady-state mRNA levels of PDGF-A and PDGF-B chain were not reduced by amiloride. In replicate EC cultures, amiloride had no effect on thrombin-stimulated monocyte adhesion. In addition, thrombin induction of PDGF production, but not monocyte adhesion, was abrogated in the absence of extracellular sodium. Thrombin stimulation of both monocyte adhesion and PDGF production appeared to involve a pertussis toxin-insensitive G protein. Thrombin induced an increase in [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to human EC membranes. GTP gamma S, in the presence of a suboptimal concentration of thrombin, caused maximal stimulation of both monocyte adhesion and PDGF production. The effect of GTP gamma S on PDGF production was at the level of transcription. These results indicate that the EC is capable of responding to a pluripotent agonist such as thrombin through multiple signaling pathways, which converge and diverge to achieve differential cellular responses.

Role of cell surface carbohydrate moieties in monocytic cell adhesion to endothelium in vitro.

Monocyte adhesion to endothelium represents the first step in the emigration of this leukocyte from blood to tissue during such pathologic and physiologic processes as atherosclerotic plaque development, wound healing, and inflammation. We have examined the role of carbohydrate moieties in the binding of mononuclear cells to endothelium in vitro. Wheat germ agglutinin (WGA) completely inhibited binding of the human monocytic cell line U937 to pig or human endothelial cells (EC). The inhibition was abolished by the presence of N-acetyl glucosamine, a preferred ligand for WGA. This sugar itself, however, had no effect on monocytic cell binding to EC, suggesting that WGA is inhibiting the cell-cell interaction by binding to a distinct sugar moiety. We tested a series of simple and phosphorylated sugars for the ability to inhibit U937 cell binding to EC. Two phosphorylated disaccharides, lactose-1-phosphate and maltose-1-phosphate, but not 14 other sugars, caused complete suppression of monocyte adhesion to EC. Among the inactive sugars were mannose-6-phosphate and fructose-1-phosphate, which have been shown by others to markedly suppress lymphocyte adhesion to EC. A nonionic detergent, n-octyl-beta-D-glucopyranoside (octyl glucoside), which contains a sugar group as a hydrophilic moiety, also inhibited U937 cell or human monocyte binding to human or porcine EC. The inhibition was observed at a nontoxic concentration of octyl glucoside and appeared to be due to an effect on the monocytic cell rather than the EC. When suboptimal doses of WGA and octyl glucoside were added in combination to the U937 cell-EC adhesion assay, the level of inhibition was greatly reduced when compared with either of the inhibitors alone, suggesting an interaction between these two blocking agents. Lactose-1-phosphate, but not octyl glucoside or WGA, blocked neutrophil adhesion to EC. In summary, our results indicate that specific cell surface carbohydrate groups are required for the adhesion of monocytes to the endothelium.

Thrombin causes increased monocytic-cell adhesion to endothelial cells through a protein kinase C-dependent pathway.

The coagulation protein thrombin has been shown to stimulate multiple endothelial-cell (EC) functions, including production of platelet-derived growth factor and of platelet-activating factor (PAF), and neutrophil adhesion. We have found that thrombin causes increased binding of monocytic cells (U937 cells and normal human monocytes) to cultured EC of various species. Maximum adhesion of monocytes to pig aortic EC occurred 6 h after thrombin treatment and remained elevated through 24 h. Stimulation of adherence by bovine alpha-thrombin was half-maximal at 15 units/ml, and reached a plateau at 50 units/ml. Catalytically inactive thrombin (phenylmethanesulphonyl fluoride-treated) had no effect on monocyte adhesion to EC. Heparin, but not the endotoxin antagonist polymyxin B, suppressed the stimulation of adhesion by thrombin without altering basal adhesion. Two lines of evidence suggested that protein kinase C (PKC) was involved in the intracellular signalling to increase monocyte adhesion to EC. First the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated monocytic-cell adhesion to EC at a dose consistent with stimulation of PKC (half-maximal response at 1-3 nM) and with a time course similar to that for thrombin stimulation (maximal by 4 h). Diacylglycerol, a physiological activator of PKC, also stimulated U937-cell adhesion to EC. Secondly, H7, a PKC inhibitor, completely blocked stimulation of monocyte adhesion to EC by thrombin or PMA. The structural analogue of H7, HA1004, which preferentially inhibits cyclic-AMP- and cyclic-GMP-dependent protein kinases, had no effect on stimulated monocyte adhesion. The PKC inhibitor also blocked the stimulation of monocyte adhesion to EC by interleukin-1 and endotoxin, but did not alter the basal level of monocyte binding to unstimulated EC. Thrombin stimulation of monocyte adhesion differed from the reported stimulation of neutrophil adhesion by thrombin in that the latter process reached a maximum in minutes rather than hours. In addition, neither PAF itself nor agents known to stimulate PAF production by EC, such as arachidonate and the Ca2+ ionophore A23187, had any effect on monocyte adhesion. These results demonstrate a PKC-dependent cytokine-like action of the coagulation protein thrombin in modulating monocytic-cell adhesion to EC, a phenomenon of potential importance in many pathological and physiological processes.

Characterization of the adhesion of the human monocytic cell line U937 to cultured endothelial cells.

Adhesion of blood-borne monocytes to the vascular endothelium is the first step in the infiltration of this leukocyte into the vessel wall or the interstitial space during inflammation. A significant role for the monocyte in both wound healing and atherogenesis is now well accepted. The molecular interactions involved in monocyte attachment to the endothelium are unknown. To study this phenomenon we have developed an in vitro system that uses the human monocytic tumor cell line U937 as a model for the blood-borne monocyte. 51Cr-labeled U937 cells were found to adhere with high affinity to cultured endothelial cells (ECs) from several sources. Much less binding was observed to either smooth muscle cells or fibroblasts from several species. Conditioned medium and cocultivation experiments ruled out the possibility that target cells could affect U937 cell binding by secretion of factors. Binding of U937 cells to porcine aortic ECs reached equilibrium after 30 min at 37 degrees C and 90 min at 4 degrees C with similar extent of binding at the two temperatures. Binding of U937 to the endothelium reached saturation at 9-12 U937 per porcine aortic EC (semi-confluent) with half-maximal binding at 1.5 X 10(6) U937 cells/ml. Bound cells dissociated with a half-life of 20 h at 37 degrees C. Adhesion of U937 cells was blocked by prior incubation of ECs with normal monocytes but not with platelets, lymphocytes, or neutrophils. Trypsin treatment or detergent solubilization of ECs inhibited U937 cell binding. A striking effect of EC density on monocytic cell adhesion was observed with bovine, rat, and porcine ECs. Confluent cultures of these cells exhibited negligible binding of U937, but when plated sparsely, the same cells were excellent targets for U937 cell adhesion. In addition, when confluent cultures of bovine aortic ECs were "wounded" with a cotton swab and then allowed to recover for 24 h at 37 degrees C, U937 cells were found to adhere most readily to the ECs migrating into the wound and neighboring the wound but not to ECs in the confluent monolayer away from the wound edge. These latter results may have implications for the focal adhesion of monocytes to the vessel wall in vivo.