The effects of calcium phosphate particles on the growth of osteoblasts

With advances in ceramics technology, calcium phosphate bioceramics have been applied as bone substitutes for several decades. The focus of this work is to elucidate the biocompatibility of the particulates of various calcium phosphate cytotoxicities. Four different kinds of calcium phosphate powders, including beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA), beta-dicalcium pyrophosphate (beta-DCP), and sintered beta-dicalcium pyrophosphate (SDCP), were tested by osteoblast cell culture. The results were analyzed by cell count, concentration of transforming growth factor-beta 1 (TGF-beta 1), alkaline phosphatase (ALP), and prostaglandin E2 (PGE2) in culture media. The changes were most significant when osteoblasts were cultured with beta-TCP and HA bioceramics. The changes in cell population of the beta-TCP and HA were quite low in the first 3 days, then increased gradually toward the seventh day. The changes in TGF-beta 1 concentration in culture medium inversely related to the changes in cell population. The ALP titer in the culture media of the beta-TCP and HA were quite high in the first 3 days, then decreased rapidly between the third and seventh days. The concentrations of PGE2 in the culture media tested were quite high on the first day, decreased rapidly to the third day, and then gradually until the seventh day. The changes in the beta-DCP and SDCP were quite similar to those of HA and beta-TCP but much less significant. We conclude that HA and beta-TCP have an inhibitory effect on the growth of osteoblasts. The inhibitins effects of the HA and beta-TCP powders on the osteoblast cell cultures possibly are mediated by the increased synthesis of PGE2.     

Excitation-calcium release uncoupling in aged single human skeletal muscle fibers

The principal objective of this communication has been to determine the manner in which two tissue culture substrata (plastic dishes and type I collagen gels) modulate the response of adult skin fibroblasts to TGF-beta 1 with respect to hyaluronan (HA) synthesis. Our results indicate that (a) fibroblasts cultured on collagen gels synthesised more HA compared to cells plated at the same density on plastic dishes, (b) this up-regulation in total HA synthesis by collagen-cultured cells was accompanied by an increase in the relative proportion of high molecular mass species of newly synthesised HA, and (c) the specific effect of TGF-beta 1 on HA synthesis was dependent upon the substratum: i.e. TGF-beta 1 inhibited HA synthesis by subconfluent fibroblasts cultured on both substrata, had no apparent effect on confluent cells cultured on collagen gels, and stimulated HA synthesis by confluent cells cultured on plastic dishes. The TGF beta-stimulated of HA synthesis by confluent fibroblasts cultured on plastic dishes persisted when these cells were transferred to collagen gels in the absence of further TGF-beta 1: interestingly, a second exposure of these plastic pre-incubated cells to TGF-beta 1 whilst growing on collagen resulted in a down-regulation in HA synthesis. Confluent fibroblasts pre-incubated with TGF-beta 1 for 24 h on plastic dishes (i.e. under conditions which stimulate HA synthesis) also displayed an HA-dependent stimulation in cell migration when subsequently plated onto collagen gels in the absence of further cytokine.     

Interstitial laser-induced thermotherapy: influence of carbonization on lesion size

Growth/differentiation factor-5 (GDF-5) is a new member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional peptide growth factors that appear to mediate many key events in cell growth and development. The effects of GDF-5 and other growth factors (epidermal growth factor, EGF; TGF-beta 1) on the proliferation of human keratinocytes and fibroblasts compared with desoximetasone and calcipotriol have been investigated. The proliferation rate was determined by a hemocytometer, MTT assay and the incorporation of [3H]-thymidine. Moreover, cell cycle analyses were performed and the influence on interleukin-1 alpha (IL-1 alpha) production in keratinocytes was measured by enzyme-linked immunosorbent assay (ELISA) because of its pronounced proinflammatory effect. In keratinocytes, GDF-5 stimulated cell proliferation to a minor extent. The drug already proved to be effective at very low concentrations (0.1 ng/ml). Growth stimulatory effects with EGF have been observed only in keratinocyte basal medium (KBM), but not in keratinocyte growth medium (KGM). TGF-beta 1 markedly inhibited the proliferation of keratinocytes at concentrations > 1 ng/ml. Calcipotriol and desoximetasone also showed a dose-dependent cell growth inhibition in epidermal cell cultures. IL-1 alpha synthesis was greatly suppressed by calcipotriol 10(-8)-10(-6) M. EGF at 10 ng/ml, in contrast, strongly stimulated IL-1 alpha production. Neither GDF-5 nor TGF-beta 1 had a significant effect on IL-1 alpha production in keratinocyte monolayer cultures. In fibroblasts, GDF-5 induced very weak antiproliferative effects. Calcipotriol and desoximetasone also inhibited cell growth in fibroblast cultures whereas proliferation and DNA synthesis were strongly stimulated by 1 ng/ml EGF. There was, however, a contradiction between TGF-beta 1 results on fibroblasts. Whereas TGF-beta 1 increased proliferation in cell number determination and in the thymidine incorporation assay, MTT assays showed slight antiproliferative effects. Due to these controversial results, in addition cell cycle analysis was employed. TGF-beta 1 led to an increased S phase, which indicates a stimulation of cell division. The different results obtained with the MTT test suggest that TGF-beta 1 may stimulate cell division of fibroblasts not only by increasing the S phase, but also by shortening the G1 phase of the cell cycle.     

Viscosupplementation: a new concept in the treatment of sacroiliac joint syndrome: a preliminary report of four cases

OBJECTIVE: To evaluate the modulatory effects of interleukin (IL)-1beta and prostaglandin (PG)E2 on the PGE2 receptor subtype EP1 in amnion cell cultures. METHODS: Amnion cell cultures were incubated in increasing concentrations of (IL)-1beta or PGE2. Cultures were also incubated in high concentrations of IL-1beta and PGE2 in combination. Changes in EP1 receptor levels were evaluated by western and northern blot analysis. Culture fluid PGE2 levels were measured by enzyme-linked immunosorbent assay. RESULTS: EP1 receptor protein levels decreased with increasing levels of PGE2 (r = -0.82, P < .05). EP1 receptor protein (r = 0.95, P < .05), EP1 mRNA (r = 0.95, P < .01), and culture fluid PGE2 levels (P < .01) were all increased after IL-1beta administration. EP1 receptor levels also increased approximately fourfold in response to IL-1beta incubation even in the presence of high agonist (PGE2) concentrations (P < .01). CONCLUSION: The results of this study show that IL-1beta might be involved in infection-induced preterm labor by interfering with the normal regulation of EP1 receptor levels and with the promotion of increased PGE2 production in amnion tissue.     

Identification of ArgBP1, an Arg protein tyrosine kinase binding protein that is the human homologue of a CNS-specific Xenopus gene

Cardiac fibroblasts constitute greater than 90% of the non-myocyte cells in the heart. Previously, it was established that cardiac fibroblasts are predisposed to transformation into a phenotype with muscle-specific features and that transforming growth factor-beta 1 (TGF-beta 1) is a specific inducer of this event. In this study the hypothesis that TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts is associated with their altered proliferative capacity is tested. Therefore the effects of TGF-beta 1 on DNA synthesis in cardiac fibroblasts under normal conditions of cell culture and in response to a potent mitogen, basic fibroblasts growth factor (bFGF) were determined. The results showed that TGF-beta 1 at 15 ng/ml (a concentration that induces fibroblast "transformation") had a regulatory effect on proliferative capacity of cardiac fibroblasts which varied as the function of cell density in culture. In subconfluent and confluent cultures, pre-treatment of cardiac fibroblasts with TGF-beta 1 for 24 h resulted in a dramatic shift in the bFGF-induced stimulation of DNA synthesis. TGF-beta 1-induced inhibition of DNA synthesis in cardiac fibroblasts coincided with their phenotypic modulation as evidenced by the expression of sarcomeric actin mRNA and morphological changes. Cross-linking studies with [125I]-labeled TGF-beta 1 showed the presence of conventional types I, II and III TGF-beta 1 receptor complexes on cardiac fibroblasts and their binding to TGF-beta 1 under the experimental conditions. In summary, these data indicate that the proliferative capacity of cardiac fibroblasts is controlled by TGF-beta 1. They further suggest that the TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts may be extended to include their altered proliferative capacity.     

No deterioration in insulin sensitivity, but impairment of both pancreatic beta-cell function and glucose sensitivity, in Japanese women with former gestational diabetes mellitus

Recent studies have demonstrated a strong correlation between infection and preterm labor. Preterm delivery is also associated with high levels of cytokines and prostaglandins in amniotic fluid. The purpose of this study was to investigate the effect of tumor necrosis factor-alpha (TNF-alpha) on the levels of cyclooxygenase, prostaglandin E2 production (PGE2), and expression of the PGE2 receptor subtype EP1 in amnion WISH cell culture. Amnion WISH cell cultures were incubated in increasing concentrations of TNF-alpha (0-50 ng/ml). Changes in cyclooxygenase and EP1 receptor proteins were evaluated by Western blot analysis. Changes in EP1 mRNA were evaluated by Northern blot, and culture fluid concentrations of PGE2 were estimated by enzyme immunoassay (EIA). EP1 protein (p<0.01), EP1 mRNA (p<0.05), cyclooxygenase-2 (COX-2) protein (p<0.001), and PGE2 concentrations (p<0.01) all increased with increasing concentrations of TNF-alpha. Changes in COX-1 protein were not observed following TNF-alpha-incubation. The results suggest that TNF-alpha may play a role in infection-induced preterm labor by its pleiotropic ability to simultaneously stimulate COX-2 activity, PGE2 concentrations, and PGE2 EP1 receptor levels in human amnion.     

Prostaglandin E2 mediates activation of hypothalamic histamine by interleukin-1beta in rats

The present study was designed to investigate the effects of peripheral interleukin-1beta (IL-1beta) on hypothalamic histamine (HA) systems. Intraperitoneal injection of IL-1beta increased the turnover rate of hypothalamic HA, which was assessed by accumulation of tele-methylhistamine after pargyline treatment. IL-1beta increased the activities of both histidine decarboxylase (HDC), an HA synthesizing enzyme, and HA-N-methyltransferase (HMT), an HA catabolizing enzyme. Pretreatment with indomethacin completely blocked the effects induced by IL-1beta on hypothalamic HA. Infusion of prostaglandin E2 (PGE2) into the third cerebroventricle increased the hypothalamic HA turnover rate, and simultaneously activated both HDC and HMT dose-dependently, but intravenous infusion of PGE2 had no effect on the dynamics of hypothalamic HA turnover. These results indicate that hypothalamic PGE2 activated by peripheral administration of IL-1beta, but not by peripheral PGE2, may enhance synthesis and release of hypothalamic HA by activation of HDC, and may facilitate degradation of extracellular histamine by activation of HMT.     

Significance of ventricular myocytes and nonmyocytes interaction during cardiocyte hypertrophy: evidence for endothelin-1 as a paracrine hypertrophic factor from cardiac nonmyocytes

BACKGROUND: In cardiac hypertrophy, both excessive enlargement of cardiac myocytes and progressive interstitial fibrosis are well known to occur simultaneously. In the present study, to investigate the interaction between ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs), mostly fibroblasts, during cardiocytes hypertrophy, we examined the change in cell size and gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cultured MCs as markers for hypertrophy in the neonatal rat ventricular cardiac cell culture system. METHODS AND RESULTS: The size of cultured MCs significantly increased in the MC-NMC coculture. Concomitantly, secretions of ANP and BNP into culture media were significantly increased in the MC-NMC coculture compared with in the MC culture (with the possible contamination of NMC <1% of MC). Moreover, in the MC culture, enlargement of MC and an increase in ANP and BNP secretions were induced by treatment with conditioned media of the NMC culture. A considerable amount of endothelin (ET)-1 production was detected in the NMC-conditioned media. BQ-123, an ET-A receptor antagonist, and bosentan, a nonselective ET receptor antagonist, significantly blocked the hypertrophic response of MCs induced by treatment with NMC-conditioned media. Angiotensin II (Ang II) (10(-10) to 10(-6) mol/L) and transforming growth factor-beta1 (TGF-beta1) (10(-13) to 10(-9) mol/L), both of which are known to be cardiac hypertrophic factors, did not induce hypertrophy in MC culture, but both Ang II and TGF-beta1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture. This hypertrophic activity of Ang II and TGF-beta1 was associated with the potentiation of ET-1 production in the MC-NMC coculture, and the effect of Ang II or TGF-beta1 on the secretions of ANP and BNP in the coculture was significantly suppressed by pretreatment with BQ-123. CONCLUSIONS: These results demonstrate that NMCs regulate MC hypertrophy at least partially via ET-1 secretion and that the interaction between MCs and NMCs plays a critical role during the process of Ang II- or TGF-beta1-induced cardiocyte hypertrophy.     

Differential development of CD4 and CD8 cytotoxic T cells (CTL) in PBMC across the leprosy spectrum; IL-6 with IFN-gamma or IL-2 generate CTL in multibacillary patients

Cation-exchange chromatography effectively concentrates the cell growth activity present in whey and we have used this process as a basis to characterise further the growth factors present in bovine milk. Under neutral conditions, total bioactivity in the growth factor-enriched cation-exchange fraction chromatographed with an apparent molecular mass of 80-100 kDa. In contrast, acid gel-filtration chromatography resolved two peaks of cell growth activity. A peak at 15-25 kDa contained the bulk of growth activity for Balb/c 3T3 fibroblasts while bio-activity for L6 myoblasts and skin fibroblasts eluted with a molecular mass of 6 kDa. A peak of inhibitory activity for Mv1Lu and MDCK cells also eluted at 15-25 kDa. Both IGF-I and IGF-II were purified from fractions that eluted at 6 kDa, although the IGF peptides alone did not account for the total bioactivity recovered. Platelet-derived growth factor (PDGF), identified by radioreceptor assay, eluted at a slightly higher molecular mass than the peak of growth activity for Balb/c 3T3 cells, and an anti-PDGF antibody was without effect on the growth of Balb/c 3T3 cells in response to the whey-derived factors. Further purification of the inhibitory activity for epithelial cells yielded a sequence for transforming growth factor beta (TGF-beta), and all inhibitory activity for Mv1Lu cells was immunoneutralised by an antibody against TGF-beta. In contrast, this antibody decreased the growth of Balb/c 3T3 fibroblasts in the whey-derived extract by only 10%. Finally, a cocktail of recombinant growth factors containing IGF-I, IGF-II, PDGF, TGF-beta and fibroblast growth factor 2 stimulated growth of Balb/c 3T3 cells to a level equivalent to only 51% of that observed in the milk-derived growth factor preparation. We conclude that: (i) cell growth activity recovered from bovine whey is present in acid-labile high molecular weight complexes; (ii) all cell growth inhibitory activity for epithelial cells can be accounted for by TGF-beta; (iii) IGF-I and IGF-II co-elute with the major peak of activity for L6 myoblasts and skin fibroblasts, although the IGF peptides alone do not explain the growth of these cells in the whey-derived extract; and (iv) neither PDGF nor TGF-beta account for the 15-25 kDa peak of Balb/c 3T3 growth activity. These data suggest the presence of additional mitogenic factors in bovine milk.     

Restoration of down-regulated PDGF receptors by TGF-beta in human embryonic fibroblasts. Enhanced response during cellular in vitro aging

The study of [125I]PDGF-BB binding to normal human embryonic lung fibroblasts, quiescent when cultured at sparsity in the presence of minute concentrations of homologous PDS, reveals approximately 2 x 10(5) binding sites for PDGF per cell; this number significantly increases during prolonged quiescence of the culture. As late as 48 h after down-regulation of PDGF receptors, the cells restore only partially their capacity to bind PDGF, with aged cells (above CPD 45) responding more rapidly and efficiently than younger ones. TGF-beta significantly enhances restoration of PDGF receptors and, in aged cells in particular, its presence results in total receptor recovery within 24 h, suggesting a concerted action of PDGF and TGF-beta regulating the proliferation of human fibroblasts in tissue regeneration.     

Hyaluronic acid metabolism in keloid fibroblasts

Hyaluronic acid (HA), an important component of the tissue extracellular matrix, is a ubiquitous glycosaminoglycan (GAG) that forms a pericellular coat on the surface of cells. It has been speculated that this pericellular HA boundary may localize cytokines, such as transforming growth factor-beta 1, which is known to stimulate collagen production. The purpose of this study was to examine the role of HA and its cell surface receptor (CD44), an active participant in HA degradation, as they relate to keloid formation. Dermal excisions from both normal patients (n = 13) and keloid patients (n = 13) were analyzed for HA content using an alcian blue staining technique. Fibroblast cell cultures were used to quantitate HA synthesis and CD44 receptor density. Histological analyses showed a greater HA content in keloid tissue compared with normal dermal tissue. In agreement with this observation, keloid fibroblasts were found to synthesize significantly more HA than normal dermal fibroblasts (2469 +/- 483 cpm versus 1122 +/- 256 cpm, P = .02). Treatment of keloid fibroblasts with triamcinolone acetonide reduced the level of HA synthesis to that of normal fibroblasts (1560 +/- 477 cpm versus 1293 +/- 264 cpm, P = .6). However, there was no significant difference in HA receptor density on keloid cells compared with normal skin fibroblasts. Therefore, the increased HA deposits found in keloids are attributable to increased synthesis rather than to decreased degradation mediated by the CD44 receptor.     

Transforming growth factor-beta1 increases mRNA levels of osteoclastogenesis inhibitory factor in osteoblastic/stromal cells and inhibits the survival of murine osteoclast-like cells

Osteoclastogenesis inhibitory factor (OCIF), also termed osteoprotegerin (OPG), is a secreted member of the tumor necrosis factor (TNF) receptor family. It inhibits bone resorption in vivo and osteoclast-like cell (OCL) formation in vitro. To better understand the biological role of OCIF, we first examined the effects of various osteotropic agents on OCIF mRNA levels in mouse calvarial osteoblasts. Northern blot analysis showed that stimulators of OCL formation such as 1,25-(OH)2D3, prostaglandin E2 (PGE2), parathyroid hormone (PTH), and interleukin 1 (IL-1) decreased OCIF mRNA levels. In contrast, transforming growth factor (TGF)-beta1 increased OCIF mRNA levels in primary osteoblasts as well as in osteoblastic/stromal cell lines. Since it was reported that both TGF-beta1 and OCIF not only inhibited OCL formation but also impaired the survival of OCL by inducing apoptosis in vitro, we next examined the possible involvement of OCIF in TGF-beta1-induced impairment of OCL survival. In a mouse bone marrow culture, we confirmed that addition of OCIF or TGF-beta1 decreased the number of surviving OCL. Anti-OCIF IgG, which completely neutralized the effect of OCIF, partially prevented the TGF-beta1-induced decrease in the number of OCL. Our results suggest that (i) downregulation of OCIF expression is one of the mechanisms for the stimulatory effects of 1,25(OH)2D3, PGE2, PTH, and IL-1 on osteoclastogenesis; and (ii) the TGF-beta1-induced apoptosis of OCL is mediated, at least in part, by upregulation of OCIF expression.     

Growth factor regulation of insulin-like growth factor (IGF) binding proteins (IGFBP) and preadipocyte differentiation in porcine stromal-vascular cell cultures

The influence of anti-IGF-1 and anti-transforming growth factor beta (TGF-beta) neutralizing antibodies on preadipocyte differentiation and secretion of IGFBPs was examined in serum free porcine stromal-vascular cultures. Cultures were stained for morphological analysis and conditioned media were collected for: TGF-beta determination by ELISA, IGF-1 by RIA, and IGFBP analysis by ligand blotting. After 6 d of treatment, anti-TGF-beta increased fat proportions by 2.7 fold compared to controls. Anti-IGF-1 decreased fat cell proportions by 14-fold. Anti-TGF-beta increased concentrations of IGF-1 5.8-fold and IGFBP-2 and IGFBP-3 by 8- and 7-fold in conditioned media whereas IGFBP-4 decreased 5-fold. Anti-IGF-1 increased concentrations of IGFBP-2 and 3 by 9- and 35-fold, respectively. TGF-beta increased concentrations of IGFBP-1, 2 and 3 by 3-fold, 18-fold and 3-fold, respectively, after 9 d in culture (6 d of treatment). There was no change in TGF-beta levels in anti-IGF-1 treated cultures compared to controls. Control antibodies and negative controls had no effect. These results provide evidence that endogenously produced IGF-1 and TGF-beta has a major influence on preadipocyte differentiation in serum free media by modulating IGFBP production/secretion.     

Status of the system of hemostasis in patients with non-insulin-dependent and insulin-dependent diabetes mellitus without clinical manifestation of vessel pathology

BACKGROUND: Murine progressive ankylosis (MPA) is a spontaneous arthropathy that produces ankylosis of peripheral and spinal joints in mice homozygous for the gene ank. This animal model bears a striking resemblance clinically, radiographically, and histologically to ankylosing spondylitis. Phosphocitrate (PC) is the only treatment known to significantly delay disease progression in MPA. Transforming growth factor-beta (TGF-beta) is important for both developmental bone formation and fracture healing, and has been detected in biopsy specimens from sacroiliac joints of patient with ankylosing spondylitis. We hypothesized that TGF-beta might be involved in the pathogenesis of MPA. METHODS: We compared the proliferative response of resting fibroblasts from normal and MPA mice to TGF-beta 1 as measured by 3H-thymidine incorporation and the effect of PC on that response. Cells were cultured with 10% serum as a positive control. The mouse fibroblast cell line, BALB/3T3, controlled for culture conditions. RESULTS: MPA and normal fibroblasts responded similarly to serum. MPA fibroblasts proliferated significantly better in TGF-beta 1 than the poorly responsive normal mouse fibroblasts. PC, at 10(-3) mol/L, inhibited the TGF-beta 1-induced proliferation of MPA and 3T3 cells, but had no effect on normal fibroblasts. CONCLUSIONS: MPA fibroblasts proliferate excessively to TGF beta 1 in vitro. This effect could be caused by altered TGF receptors, changes in signal transduction, or impaired inhibition of the TGF-beta signal. This excessive response is blocked by PC. These results give further clues as to how PC inhibits the progression of ankylosis in MPA.     

Use of aquatic organisms as models to determine the in vivo contribution of flavin-containing monooxygenases in xenobiotic biotransformation

Human corneal fibroblasts (HCF) inhibit T cell alloresponse in mixed leukocyte response-human corneal fibroblast coculture. The inhibition is contact independent, insensitive to indomethacin, and is enhanced by pretreatment of HCF with interferon-gamma (IFN-gamma). To investigate cytokine-dependent mechanisms of inhibition of T cell alloresponse by HCF, the capacity of cultured HCF to produce transforming growth factor-beta (TGF-beta) and the modulatory role of IFN-gamma on their TGF-beta production were investigated by radioreceptor binding inhibition assay (RRA) and the standard mink cell bioassay (BIA). The net total TGF-beta concentration of 4 day culture supernatants from IFN-gamma-treated HCF, measured by RRA, was 11.5 ng/ml. The net total bioactive TGF-beta concentrations of 4 day culture supernatants from HCF, before and after treatment with IFN-gamma, measured by BIA, were 2.0 and 4.8 ng/ml, respectively. These findings indicate that HCF produce TGF-beta and increase their TGF-beta output under the influence of the proinflammatory cytokine IFN-gamma. Media-borne TGF-beta binding proteins appeared to be primarily responsible for the discrepancy between the TGF-beta values measured by RRA and BIA. Active exclusion of TGF-beta binding proteins from intraocular fluids may have an important role in the maintenance of TGF-beta-dependent ocular immune privilege. Corneal fibroblasts may utilize TGF-beta-dependent mechanisms to maintain the immunosecluded environment of the cornea and to preserve the homeostasis of corneal optical competency. Interferon-gamma may enhance corneal immunoseclusion by upregulating the TGF-beta output of the corneal fibroblasts.     

Enhancement of ovine trophoblast interferon by granulocyte macrophage-colony stimulating factor: possible involvement of protein kinase C

In this study, we examined the effects of age on collagen and total protein synthesis by ligament fibroblasts in response to growth factors. Three different doses of transforming growth factor-beta 1 (TGF-beta 1) or epidermal growth factor (EGF) were individually added to in vitro fibroblast cultures from the medial collateral ligament (MCL) of skeletally immature (age 3 months), mature (age 12 months) and senescent (age 48-51 months) rabbits. Analysis of the effects of age revealed that fibroblasts from senescent rabbits produced significantly less collagen in response to TGF-beta 1 or EGF stimulation when compared to fibroblasts from immature rabbits. Furthermore, increased age was found to result in significant reductions in the baseline levels of collagen synthesis but not total protein synthesis. Additionally, collagen and total protein synthesis by MCL fibroblasts were significantly affected by the TFG-beta 1 dose, but not by the EGF dose. When fibroblasts were normalized to their own controls, the increase in collagen and total protein synthesis due to TGF-beta 1 and EGF for the senescent group were found to be greater than those for the skeletally immature rabbits at all doses. This demonstrates that MCL fibroblasts from senescent rabbits are responsive to growth factors.     

Modulation of transforming growth factor-beta 1 effects by cytokines

The purpose of the present study was to determine the effects of human recombinant transforming growth factor-beta 1 (TGF-beta 1) on the proliferation of normal cell and cancer cell lines and to evaluate the mechanism of TGF-beta-induced immunosuppression. Murine H238 fibrosarcoma and human UC-11 glioblastoma cells showed no proliferative change in the presence of TGF-beta, whereas the growth of human LS174T colon adenocarcinoma cells was significantly enhanced at the lower concentrations of TGF-beta. In contrast, Mono/Mac-6, a human monocyte cell line, human peripheral blood mononuclear (PBMN) cells, and BALB/c mouse spleen cells were significantly suppressed by 2.5 to 250 ng/ml of TGF-beta. In order to investigate the mode of action, TGF-beta and other cytokines were added 0, 1, and 2 days after initiation of the culture. Mono/Mac-6 cells showed that 2 days are needed for TGF-beta-induced suppression. Simultaneous addition of TGF-beta and tumor necrosis-alpha (TNF-alpha; 600 units/ml) to Mono/Mac-6 cells resulted in nearly complete suppression by day 3. IL-2, and to a lesser extent IL-4, was able to counteract the suppressive effects of TGF-beta on mitogen-stimulated spleen cells. However, our results indicate that IL-2 is not as effective in restoring responsiveness once T cell activation is well underway. IL-1 and interferon-gamma had no effects on TGF-beta-mediated immunosuppression. Since TGF-beta depressed normal cell growth and since IL-2 could effectively counteract the suppression, we assayed for IL-2 production. When normal spleen cells were treated with 2.5 ng of TGF-beta/ml, a 3.4-fold decrease in IL-2 production was observed. This is a potential mechanism for TGF-beta-mediated immunosuppression.