Kinetics of expression of connective tissue growth factor gene during liver regeneration after partial hepatectomy and D-galactosamine-induced liver injury in rats

Connective tissue growth factor (CTGF) is up-regulated by TGF-beta1 during wound healing. The present study examined the expression of CTGF during regeneration after 70% partial hepatectomy (PH) or d-galactosamine (GalN)-injured liver in rats. CTGF, TGF-beta1, and type I collagen mRNAs were semiquantified by a ribonuclease protection assay. After PH, TGF-beta1 and type I collagen were increased at 2-6 h and at 12-48 h. CTGF increased at 6 h and returned to the control level thereafter. The ribonuclease protection assay of cultured hepatic stellate cells (HSC) and in situ hybridization suggest that the cells express CTGF along sinusoid might be HSCs. After GalN administration, CTGF increased at 2-96 h with a shoulder peak at 6-12 h followed by a main peak at 24 h. TGF-beta1 and type I collagen were up-regulated with kinetics similar to those of CTGF. The different kinetics between PH and GalN regenerations indicate that regulation of CTGF in the two processes is different. Higher TGF-beta1 expression after inflammatory/necrotic process in the GalN regeneration may caused the prolonged CTGF expression.     

Regulation of TGF-beta 1-induced connective tissue growth factor expression in airway smooth muscle cells

Transforming growth factor (TGF)-beta may play an important role in airway remodeling, and the fibrogenic effect of TGF-beta may be mediated through connective tissue growth factor (CTGF) release. We investigated the role of MAPKs and phosphatidylinositol 3-kinase (PI3K) and the effects of inflammatory cytokines on TGF-beta-induced CTGF expression in human airway smooth muscle cells (ASMC). We examined whether Smad signal was involved in the regulatory mechanisms. TGF-beta 1 induced a time- and concentration-dependent expression of CTGF gene and protein as analyzed by real-time RT-PCR and Western blot. Inhibition of ERK and c-jun NH(2)-terminal kinase (JNK), but not of p38 MAPK and PI3K, blocked the effect of TGF-beta 1 on CTGF mRNA and protein expression and on Smad2/3 phosphorylation. T helper lymphocyte 2-derived cytokines, IL-4 and IL-13, attenuated TGF-beta 1-stimulated mRNA and protein expression of CTGF and inhibited TGF-beta 1-stimulated ERK1/2 and Smad2/3 activation in ASMC. The proinflammatory cytokines tumor necrosis factor-alpha and IL-1 beta reduced TGF-beta 1-stimulated mRNA expression of CTGF but did not inhibit TGF-beta-induced Smad2/3 phosphorylation. TGF-beta 1-stimulated CTGF expression is mediated by mechanisms involving ERK and JNK pathways and is downregulated by IL-4 and IL-13 through modulation of Smad and ERK signals.     

MMP-3 provokes CTGF/CCN2 production independently of protease activity and dependently on dynamin-related endocytosis, which contributes to human dental pulp cell migration

Matrix metalloproteinase-3 (MMP-3) expression is promoted after pulpotomy, and application of MMP-3 to dental pulp after pulpotomy accelerates angiogenesis and hard tissue formation. However, the mechanism by which MMP-3 promotes dental pulp wound healing is still unclear. Connective tissue growth factor/CCN family 2 (CTGF/CCN2), a protein belonging to the CCN family, is considered to participate in wound healing, angiogenesis, and cell migration. In this study, we examined the involvement of CTGF/CCN2 in MMP-3-induced cell migration in human dental pulp (fibroblast-like) cells. In human dental pulp cells, MMP-3 promoted cell migration, but this effect was clearly blocked in the presence of anti-CTGF/CCN2 antibody. MMP-3 provoked mRNA and protein expression and secretion of CTGF/CCN2 in a concentration- and time-dependent manner. The MMP-3 inhibitor NNGH failed to suppress MMP-3-induced CTGF/CCN2 protein expression. The potent dynamin inhibitor dynasore clearly inhibited MMP-3-induced CTGF/CCN2 expression. These results strongly suggest that MMP-3 induces CTGF/CCN2 production independently of the protease activity of MMP-3 and dependently on dynamin-related endocytosis, which is involved in cell migration in human dental pulp cells.     

Down-regulation of IL-2 receptor α (CD25) characterizes human γδ-T cells rendered resistant to apoptosis after CD2 engagement in the presence of IL-12

We recently identified a CD2-mediated, IL-12-dependent signaling pathway that inhibits apoptosis in mitogen-stimulated human gammadelta-T cells. Here we show that gammadelta-T cells which acquire resistance to mitogen-induced apoptosis upregulate IL-12 receptor beta 1 subunit (IL-12Rbeta1); in contrast, gammadelta-T cells which remain sensitive to mitogen-induced apoptosis fail to express IL-12Rbeta1. Next we show that gammadelta-T cells which are rendered resistant to mitogen-induced apoptosis attenuate their expression of the IL-2 receptor alpha chain (IL-2Ralpha/CD25), this in part accounting for their acquired resistance to IL-2-induced death. In contrast, apoptosis-sensitive gammadelta-T cells are shown to persist in their expression of IL-2Ralpha/CD25, thus remaining sensitive to IL-2-induced death. Moreover, we show that apoptosis-resistant, but not apoptosis-sensitive, gammadelta-T cells display an enhanced responsiveness to IL-15, a finding in keeping with the known function of IL-15 as a growth and survival factor. Finally, we present evidence to suggest that this differential responsiveness to IL-15 occurs in part by the increased expression of the IL-15Ralpha chain on apoptosis-resistant gammadelta-T cells, compared to apoptosis-sensitive gammadelta-T cells. The biological and clinical implications of these findings are discussed.     

Endoglin expression regulates basal and TGF-beta1-induced extracellular matrix synthesis in cultured L6E9 myoblasts.

BACKGROUND/AIMS: TGF-beta1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-beta) receptor complex. Endoglin is upregulated by TGF-beta1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-beta1. METHODS: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. RESULTS: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased alpha2 (I) procollagen mRNA expression in endoglin transfectants. TGF-beta1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. CONCLUSION: These results demonstrate that endoglin expression negatively regulates basal and TGF-beta1-induced CTGF and collagen expression and synthesis.     

Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair.

Connective tissue growth factor (CTGF) is a cysteine-rich peptide that exhibits platelet-derived growth factor (PDGF)-like biological and immunological activities. CTGF is a member of a family of peptides that include serum-induced immediate early gene products, a v-src-induced peptide, and a putative avian transforming gene, nov. In the present study, we demonstrate that human foreskin fibroblasts produce high levels of CTGF mRNA and protein after activation with transforming growth factor beta (TGF-beta) but not other growth factors including PDGF, epidermal growth factor, and basic fibroblast growth factor. Because of the high level selective induction of CTGF by TGF-beta, it appears that CTGF is a major autocrine growth factor produced by TGF-beta-treated human skin fibroblasts. Cycloheximide did not block the large TGF-beta stimulation of CTGF gene expression, indicating that it is directly regulated by TGF-beta. Similar regulatory mechanisms appear to function in vivo during wound repair where there is a coordinate expression of TGF-beta 1 before CTGF in regenerating tissue, suggesting a cascade process for control of tissue regeneration and repair.     

Tumor necrosis factor alpha suppresses the induction of connective tissue growth factor by transforming growth factor-beta in normal and scleroderma fibroblasts

Connective tissue growth factor (CTGF) is overexpressed in a variety of fibrotic disorders, presumably secondary to the activation and production of transforming growth factor-beta (TGF-beta), a key inducer of fibroblast proliferation and matrix synthesis. The CTGF gene promoter has a TGF-beta response element that regulates its expression in fibroblasts but not epithelial cells or lymphocytes. Recent studies have shown that the macrophage-produced cytokine tumor necrosis factor alpha (TNFalpha) is necessary to promote inflammation and to induce genes, such as matrix metalloproteinases, involved with the early stages of wound healing. In this study, we examined the ability of TNFalpha to modulate CTGF gene expression. TNFalpha was found to suppress the TGF-beta-induced expression of CTGF protein in cultured normal fibroblasts. The activity of TNFalpha was blocked by NF-kappaB inhibitors. We showed that sequences between -244 and -166 of the CTGF promoter were necessary for both TGF-beta and TNFalpha to modulate CTGF expression. There was a constitutive expression of CTGF by scleroderma fibroblasts that was increased by TGF-beta treatment. Although TNFalpha was able to repress TGF-beta-induced CTGF and collagen synthesis both in normal and scleroderma skin fibroblasts, fibroblasts cultured from scleroderma patients were more resistant to TNFalpha as TNFalpha was unable to suppress the basal level of CTGF expression in scleroderma fibroblasts. Thus, we suspect that the high level of constitutive CTGF expression in scleroderma fibroblasts and its inability to respond to negative regulatory cytokines may contribute to the excessive scarring of skin and internal organs in patients with scleroderma.     

The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process

Several lines of in vitro evidence suggest the potential role of IFN-gamma in angiogenesis and collagen deposition, two crucial steps in the wound healing process. In this report, we examined the role of IFN-gamma in the skin wound healing process utilizing WT and IFN-gamma KO mice. In WT mice, excisional wounding induced IFN-gamma mRNA and protein expression by infiltrating macrophages and T cells, with a concomitant enhancement of IL-12 and IL-18 gene expression. Compared with WT mice, IFN-gamma KO mice exhibited an accelerated wound healing as evidenced by rapid wound closure and granulation tissue formation. Moreover, IFN-gamma KO mice exhibited enhanced angiogenesis with augmented vascular endothelial growth factor mRNA expression in wound sites, compared with WT mice, despite a reduction in the infiltrating neutrophils, macrophages, and T cells. IFN-gamma KO mice also exhibited accelerated collagen deposition with enhanced production of TGF-beta1 protein in wound sites, compared with WT mice. Furthermore, the absence of IFN-gamma augmented the TGF-beta1-mediated signaling pathway, as evidenced by increases in the levels of total and phosphorylated Smad2 and a reciprocal decrease in the levels of Smad7. These results demonstrate that there is crosstalk between the IFN-gamma/Stat1 and TGF-beta1/Smad signaling pathways in the wound healing process.     

Regulation of lymphokine-activated killer activity and pore-forming protein gene expression in human peripheral blood CD8+ T lymphocytes. Inhibition by transforming growth factor-beta.

The effect of transforming growth factor-beta 1 (TGF-beta) on activation-induced CD8+ T cell cytotoxicity and gene expression was investigated. TGF-beta was demonstrated to inhibit pore-forming protein (PFP) mRNA expression and total benzoyloxycarbonyl-L-lysine thiobenzyl ester esterase activity in CD8+ T cells cultured with IL-2 and OKT3 mAb for 6 to 18 days. Consistently, in the absence or presence of TGF-beta, the PFP mRNA expression and lymphokine-activated killer (LAK) activity of CD8+ T cells were closely correlated. The inhibitory effects of TGF-beta on both CD8+ T cell PFP mRNA expression and LAK activity were reversible by removal of TGF-beta from the culture. Expression of lymphokines, adhesion/recognition molecules, and activated p55 IL-2R, previously implicated in the lytic mechanism of cytotoxic lymphocytes, either was not detectable or did not correlate with TGF-beta inhibition of LAK activity. In addition, independently of effector/target cell binding, the lectin- or heteroconjugated antibody-dependent cellular cytotoxicity of IL-2/OKT3 mAb-activated CD8+ T cells was inhibited by preculture with TGF-beta. TGF-beta also inhibited the rapid activation-induced expression of PFP mRNA and cytotoxic potential in resting T cells, thereby indicating that the effect of TGF-beta was independent of T cell proliferation. TGF-beta inhibition of CD8+ T cell PFP mRNA expression and cytotoxic potential was TGF-beta dose dependent; however, a variety of activation stimuli (including IL-2, IL-6, and OKT3 mAb) were all similarly inhibited by TGF-beta. Therefore, TGF-beta may be an important general regulator of CD8+ T cell cytotoxic function, in particular by suppressing expression of PFP, a major cytolytic protein implicated in the lytic function of cytotoxic lymphocytes.     

TGF-beta inhibits proliferation of and promotes differentiation of human promonocytic leukemia cells.

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in a variety of responses associated with wound healing and inflammation. Thus, TGF-beta 1 enhances production of several extracellular matrix proteins both in vitro and in vivo, is chemotactic for monocytes, and alters the functioning of lymphocytes. We have examined the ability of TGF-beta 1 to affect the behavior of human THP-1 promonocytic leukemia cells, a cell line with the capacity to differentiate into macrophage-like cells. TGF-beta 1 reduces the growth rate of these cells, induces morphologic changes, and promotes adherence to culture surfaces. In addition, the adherent cell population expresses high levels of esterase activity, acquires the ability to ingest latex beads, and releases elevated levels of interleukin 1. TGF-beta 1-treated cells also express elevated levels of the beta 2 family of integrins. Taken together, these results suggest that TGF-beta 1 is capable of promoting the maturation of promonocytic cells into macrophages. This outcome has implications at wound sites where TGF-beta 1 and a myriad of other factors interact with many cell types to facilitate healing.     

Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.     

Predominant Th2/Tc2 polarity of tumor-infiltrating lymphocytes in human cervical cancer

Cytotoxic T lymphocytes (Tc) play a central role in cellular immunity against cancers. The cytotoxic potential of freshly isolated tumor-infiltrating lymphocytes (TILs) is usually not expressed. This suggests the possible existence of as yet unspecified and perhaps complex immunosuppressive factors or cytokines that affect the anti-tumor capacity of these TILs in the tumor milieu. In the present study, we demonstrated for the first time that TILs derived from human cervical cancer tissue consist mainly of Th2/Tc2 phenotypes. In vitro kinetic assays further revealed that cancer cells could direct the tumor-encountered T cells toward the Th2/Tc2 polarity. Cancer cells promote the production of IL-4 and down-regulate the production of IFN-gamma in cancer-encountered T cells. The regulatory effects of cervical cancer cells are mediated mainly by IL-10, and TGF-beta plays only a synergistic role. The cancer-derived effects can be reversed by neutralizing anti-IL-10 and anti-TGF-beta Abs. IL-10 and TGF-beta are present in cancer tissue and weakly expressed in precancerous tissue, but not in normal cervical epithelial cells. Our study strongly suggests important regulatory roles of IL-10 and TGF-beta in cancer-mediated immunosuppression.     

Transforming growth factor-beta 1 and IL-4 regulate the adhesiveness of Peyer's patch high endothelial venule cells for lymphocytes.

The adhesion of lymphocytes to endothelial cells lining the postcapillary high endothelial venules (HEV) is the first step in their emigration from the bloodstream into lymph nodes and Peyer's patches (PP). We have recently shown that the adhesiveness of cultured rat lymph node and PP HEV cells for thoracic duct lymphocytes can be increased significantly by pretreatment with TNF-alpha, IFN-gamma, and IL-4. In the present study we investigated the role of transforming growth factor-beta 1 (TGF-beta) on the adhesiveness of nonstimulated and cytokine-stimulated PP HEV cells for rat lymphocytes. The results indicated that at picomolar concentrations, TGF-beta significantly (p less than 0.001) decreased the ability of PP HEV cells to adhere 51Cr-labeled rat lymphocytes. Maximal inhibition was observed with a TGF-beta dose of 0.5 ng/ml and an incubation time of 6 to 12 h. TGF-beta did not affect the morphology of HEV cells and had no adverse effect on their viability. Moreover, the decrease in HEV adhesiveness by TGF-beta was reversible, with lymphocyte binding returning to control level 24 h after removal of the cytokine. The specificity of TGF-beta was confirmed by the ability of neutralizing anti-TGF-beta 1 antibody, but not control serum, to abolish the inhibitory properties of the cytokine. In addition, TGF-beta completely abrogated the increased adhesiveness of PP HEV cells normally induced by TNF-alpha or IFN-gamma. In contrast, TGF-beta had no effect on the stimulating effects of IL-4. Moreover, preincubation of PP HEV cells with TGF-beta did not alter the ability of these cells to respond to IL-4. Importantly, the adhesion of rat lymphocytes to IL-4-stimulated PP HEV cells can be blocked by pretreatment of lymphocytes with the PP-homing receptor-specific 1B.2.6 antibody whereas pretreatment of human mononuclear cells with anti-very late activation antigen-4 alpha antibody inhibited only partially the binding of these cells to the IL-4-stimulated PP HEV monolayers. Taken together, these findings strongly suggest that TGF-beta and IL-4 play important regulatory roles in lymphocyte-HEV adhesion and that the stimulatory effect of IL-4 is mediated at least in part through the increased expression of organ-specific ligands on HEV cells.     

Aberrant Wound Healing in an Epidermal Interleukin-4 Transgenic Mouse Model of Atopic Dermatitis

Wound healing in a pre-existing Th2-dominated skin milieu was assessed by using an epidermal specific interleukin-4 (IL-4) transgenic (Tg) mouse model, which develops a pruritic inflammatory skin condition resembling human atopic dermatitis. Our results demonstrated that IL-4 Tg mice had delayed wound closure and re-epithelialization even though these mice exhibited higher degrees of epithelial cell proliferation. Wounds in IL-4 Tg mice also showed a marked enhancement in expression of inflammatory cytokines/chemokines, elevated infiltration of inflammatory cells including neutrophils, macrophages, CD3+ lymphocytes, and epidermal dendritic T lymphocytes. In addition, these mice exhibited a significantly higher level of angiogenesis as compared to wild type mice. Furthermore, wounds in IL-4 Tg mice presented with larger amounts of granulation tissue, but had less expression and deposition of collagen. Taken together, an inflamed skin condition induced by IL-4 has a pronounced negative influence on the healing process. Understanding more about the pathogenesis of wound healing in a Th2- dominated environment may help investigators explore new potential therapeutic strategies.     

Gamma c-signaling cytokines induce a regulatory T cell phenotype in malignant CD4+ T lymphocytes

In this study, we demonstrate that malignant mature CD4(+) T lymphocytes derived from cutaneous T cell lymphomas (CTCL) variably display some aspects of the T regulatory phenotype. Whereas seven cell lines representing a spectrum of primary cutaneous T cell lymphoproliferative disorders expressed CD25 and TGF-beta, the expression of FOXP3 and, to a lesser degree, IL-10 was restricted to two CTCL cell lines that are dependent on exogenous IL-2. IL-2, IL-15, and IL-21, all of which signals through receptors containing the common gamma chain, induced expression of IL-10 in the IL-2-dependent cell lines as well as primary leukemic CTCL cells. However, only IL-2 and IL-15, but not IL-21, induced expression of FOXP3. The IL-2-triggered induction of IL-10 and FOXP3 expression occurred by signaling through STAT3 and STAT5, respectively. Immunohistochemical analysis of the CTCL tissues revealed that FOXP3-expressing cells were common among the CD7-negative enlarged atypical and small lymphocytes at the early skin patch and plaque stages. Their frequency was profoundly diminished at the tumor stage and in the CTCL lymph node lesions with or without large cell transformation. These results indicate that the T regulatory cell features are induced in CTCL T cells by common gamma chain signaling cytokines such as IL-2 and do not represent a fully predetermined, constitutive phenotype independent of the local environmental stimuli to which these malignant mature CD4(+) T cells become exposed.