Tumor necrosis factor and IL-1 in New Zealand Black/White mice. Enhanced gene expression and acceleration of renal injury

TNF and IL-1 are potent immunologic and inflammatory cytokines. We have previously reported increased levels of mRNA for TNF alpha and IL-1 beta in MRL-lpr mice with lupus nephritis. To determine whether the increased levels of TNF and IL-1 mRNA are a more general feature of mice with lupus nephritis we studied cytokine gene expression in female NZB x NZW F1 (NZB/W) mice by Northern blot analysis. Enhanced steady state levels of mRNA for TNF alpha and IL-1 beta, but not IL-1 alpha, were detected in the renal cortices of animals with lupus nephritis. To determine whether administration of TNF or IL-1 would accelerate renal injury and mortality, we injected murine rTNF alpha or rIL-1 alpha i.p. into female NZB/W or C3H/FeJ mice at two doses, 2.0 micrograms or 0.2 micrograms, three times weekly for 2 or 4 mo beginning at 2 or 4 mo of age. Administration of the lower dose of each cytokine accelerated renal disease and mortality rate when treatment was initiated at 4 mo of age. At the higher dose, neither cytokine promoted disease. Treatment administered from 2-4 mo of age did not accelerate renal disease. This observation suggests that in order to cause renal injury, these cytokines must interact with other pathologic features present in these animals after 4 mo of age. These findings support the hypothesis that TNF and IL-1 can contribute to nephritis in murine models of lupus. Taken together with previously published data, we propose that TNF and IL-1 have differential dose effects on renal disease. The dose of TNF and IL-1 and the stage of disease activity dictate the pathogenic action of these cytokines.     

Transforming growth factor-beta (TGF beta) is chemotactic for human monocytes and induces their expression of angiogenic activity

TGF beta stimulates human blood monocyte migration, with peak migratory response occurring consistently at a concentration of 16-100 fg/ml. Checkerboard analysis revealed both chemotactic and chemokinetic components to this response. At higher concentrations (10-100 pg/ml), TGF beta stimulated expression of angiogenic activity by monocytes. While mRNA for TNF alpha was undetectable in resting monocytes, high steady state levels of TNF alpha mRNA were rapidly induced in TGF beta-treated monocytes. TGF beta is secreted by a number of neoplastic cells as well as normal cells such as platelets and lymphocytes. TGF beta may recruit monocytes from the circulation, and subsequently activate them to express angiogenic activities such as TNF alpha, thus playing an important role in wound repair, inflammation and tumor growth.     

Antisense inhibition of c-myc expression reveals common and distinct mechanisms of growth inhibition by TGF beta and TNF alpha

Downregulation of the c-myc gene in HL-60 cells is associated with growth inhibition and induction of differentiation. Previous studies have reported that the growth inhibitors TGF beta and TNF alpha downregulate c-myc mRNA levels, suggesting the possibility that these agents may exert some of their phenotypic effects via c-myc downregulation. Our study demonstrates that although both growth inhibitors produce a similar decrease in c-myc protein synthesis, TNF alpha produces a greater growth inhibition and differentiation induction in HL-60 cells. Combined addition of anti-myc oligomer with either growth inhibitor produces no additive effect. In fact, 4 microM anti-myc oligomer produces the same growth and differentiation effects as does 10 ng/ml TGF beta 1. We conclude that downregulation of c-myc expression represents a common mechanism of growth inhibition by TGF beta and TNF alpha, but that TNF alpha possesses an additional effect that is independent of c-myc expression.     

Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor beta is altered by cellular differentiation

Changes in epithelial substrate have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGF beta 1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen alpha 1 (IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pM TGF beta 1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1-3 pM TGF beta 1. Incremental doses of TGF beta 1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGF beta 1 induced the expression of fibronectin mRNA to levels seen in TGF beta-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen alpha 1 (IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGF beta induction of collagen alpha 1 (IV) mRNA was signaled by proteins already present in the cells but that TGF beta required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGF beta action in regulating reepithelialization.     

Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitary-derived cells.

Transforming growth factor-beta 1 (TGF beta 1) is a multifunctional regulator of cell growth and differentiation. We report here that TGF beta 1 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGF alpha and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF beta 1 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGF alpha mRNA level. The effect of TGF beta 1 on TGF alpha mRNA down-regulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF beta 1) and time dependent (minimum of 2-day treatment with TGF beta 1 was required before a decrease in TGF alpha mRNA was observed). Studies on TGF alpha mRNA stability indicated that TGF beta 1 did not alter the TGF alpha mRNA half-life. Treatment of the TGF beta 1 down-regulated cells with EGF resulted in the stimulation of TGF alpha mRNA levels; thus, the TGF beta 1-treated cells remained responsive to EGF. The decreased proliferation in response to TGF beta 1 could be only partially reversed by simultaneous treatment of the cells with EGF (10(-9)M) and TGF beta 1 (3.0 ng/ml). Qualitatively, the TGF beta 1-induced reduction of TGF alpha mRNA content was independent of cell density. TGF beta 1 treatment of the anterior pituitary-derived cells also reduced the levels of c-myc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGF alpha synthesis by a polypeptide growth factor and suggest that TGF beta 1 may be a physiological regulator of TGF alpha production in vivo.     

Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease

The liver is a major target of injury in obese patients. Non-alcoholic fatty liver disease (NAFLD) is present in 60-90% of obese Americans and can range from simple steatosis to the more severe non-alcoholic steatohepatitis (NASH). The onset of a chronic inflammatory reaction marks the progression from simple steatosis to NASH and the expansion of adipose tissue is strongly associated with angiogenesis. Therefore, we determined the serum concentration of inflammatory [tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6)] and angiogenic [vascular endothelial growth factor A (VEGF)] cytokines and soluble VEGF receptors 1 and 2 (sVEGFR1, sVEGFR2) in the serum of an obese population with simple steatosis and NASH compared to healthy controls. Moreover, we determined the TNFα, IL6, VEGF, VEGFR1 and VEGFR2 gene expression in the liver of these simple steatosis and NASH patients. The population consisted of 30 obese patients, which were diagnosed with simple steatosis and 32 patients with NASH and compared to 30 age-and-sex matched healthy controls. Mean serum TNFα levels were elevated in the serum of simple steatosis and NASH patients compared to healthy controls, reaching significance in NASH patients. IL6 was significantly increased in simple steatosis and NASH patients compared to the healthy controls. VEGF levels were significantly elevated in patients with simple steatosis and borderline significantly elevated in NASH patients compared to the serum levels of healthy control subjects. The concentration of sVEGFR1 was significantly increased in serum of simple steatosis and NASH patients compared to controls. sVEGFR2 concentration was not significantly different in the three groups. TNFα mRNA expression was higher in NASH patients compared to simple steatosis patients. Hepatic gene expression of VEGF, VEGFR1 and VEGFR2 were slightly decreased in NASH patients compared to simple steatosis patients. These data indicate the involvement of inflammatory (TNFα and IL6), angiogenic (VEGF) cytokines and sVEGFR1 in the pathophysiology of NAFLD.     

Follicular stage-dependent tumor necrosis factor alpha-induced hen granulosa cell integrin production and survival in the presence of transforming growth factor alpha in vitro

The link between cell adhesion to extracellular matrix and integrin-mediated survival signals has been established in several physiological systems, and roles for the cytokines tumor necrosis factor alpha (TNF alpha) and transforming growth factor alpha (TGF alpha) have been suggested. TGF alpha stimulates fibronectin production in hen granulosa cells and is an important survival factor during follicular maturation. In contrast, the role of TNF alpha and its possible interaction with TGF alpha in the regulation of granulosa cell fate (death versus survival) during ovarian follicular development have not been fully elucidated. The object of the current study was to determine if TNF alpha and TGF alpha interact in the regulation of hen granulosa cell fibronectin and integrin content in the context of cell death and survival during follicular development. TGF alpha (0.1 or 10 ng/ml), but not TNF alpha (0.1 or 10 ng/ml), increased both cellular and secreted fibronectin content in granulosa cell cultures of F5,6 but not F1 follicles. The expression of integrin beta(3) subunit was also stimulated by TGF alpha in a follicular stage-dependent manner, and culture of F5,6 granulosa cells with TNF alpha in the presence of maximal stimulatory concentrations of TGF alpha potentiated this response. TGF alpha increased both F5,6 and F1 granulosa cell [(3)H]thymidine incorporation but not 3-(4,5-dimethylthiazol-2-yl)3,5-diphenyl tetrazolium bromide (MTT) metabolism. Although TNF alpha had no effect on [(3)H]thymidine incorporation irrespective of the presence of the growth factor, MTT metabolism was higher in F5,6 granulosa cells cultured for 24 h with both TNF alpha and TGF alpha than with either cytokine alone. Incubation of F5,6 granulosa cells for 48 and 72 h resulted in a TGF alpha-inhibited loss of cellular adhesion and detachment of granulosa cells from the growth surface. Although TNF alpha alone had no effect on cell morphology, it facilitated the reorganization of the granulosa cells into multicellular follicle-like structures in the presence of the growth factor. DNA degradation significantly increased between 0 and 72 h of culture in the absence of the cytokine but was suppressed by the addition of TGF alpha but not of TNF alpha. However, fluorometric analysis indicated that the primary type of cell death exhibited by F5,6 granulosa cells during extended culture and attenuated by the presence of TNF alpha and TGF alpha was necrosis and not apoptosis. The current study demonstrates that TNF alpha and TGF alpha interact in the regulation of granulosa cell integrin content and cell survival in vitro in a follicular stage-dependent manner. These findings suggest that follicular development is accompanied by a change in the intraovarian role of TNF alpha; it is atretogenic prior to follicular selection but prevents follicular demise during preovulatory growth.     

Increased mRNA expression and protein secretion of interleukin-6 in primary human osteoblasts differentiated in vitro from rheumatoid and osteoarthritic bone

We have investigated the expression and synthesis of potential bone-resorbing cytokines, interleukin-6 (IL-6), interleukin-1 (IL-1), and tumor necrosis factor (TNF) in rheumatoid arthritic (RA) and osteoarthritic (OA) bone, two common diseases which are associated with bone loss. Primary human osteoblast (hOB) cultures were established to determine the temporal mRNA expression of IL-6, IL-1 (alpha and beta), and TNF (alpha and beta) in relation to osteoblast growth and phenotypic genes. IL-6 mRNA levels were found to be significantly higher (P < 0.04) in both OA hOB (17 patients) and RA hOB (10 patients) compared to normal (NO) hOB (9 patients) and reached five-fold increases in OA hOB and 13-fold increases in RA hOB. Maximal levels of IL-6 are expressed at Day 21 which corresponds to the mineralization stage reflected by decreasing collagen I (alpha(1)), osteopontin, bone sialoprotein, alkaline phosphatase mRNA levels, while osteocalcin (OC) mRNA levels increased. IL-6 protein levels also were significantly higher (P < 0.05) in OA hOB and RA hOB compared to NO hOB. These increases were not attributable to sex or age of the donor bone. Neither the mRNA encoding IL-1(alpha and beta) and TNF(alpha and beta) nor the related proteins were detectable. These results indicate that differentiated OA hOB and RA hOB within a bone tissue-like matrix constitutively express and secrete high levels of IL-6. This inherent property suggests that these osteoblasts, independent of local inflammatory parameters, can contribute to enhanced recruitment of osteoclast progenitors and thereby bone resorption.     

In vitro production of cytokines is influenced by sulfatide and its precursor galactosylceramide.

Effects of sulfatide and its precursor galactosylceramide (gal-cer) on the kinetics of production of cytokines were studied. In human mononuclear leucocytes, gal-cer but not sulfatide induced significantly increased amounts of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF) mRNA. In phytohemagglutinin-stimulated cultures, gal-cer increased the levels of IL-1beta and IL-6 mRNA and secreted IL-1beta and IL-6, while sulfatide decreased the amounts of IL-6 mRNA and secreted IL-6. Gal-cer also increased TNF secretion. In lipopolysaccharide-stimulated cells, sulfatide but not gal-cer decreased the secretion of IL-1beta and IL-10, a potent suppressor of production of many cytokines. Thus, sulfatide and gal-cer affect cytokine production differently, most likely at the level of gene expression. This may have implications in diseases where inflammatory cytokines play a pathogenic role.     

8-Chloro-cAMP inhibits transforming growth factor alpha transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation.

Differential regulation of the regulatory subunits of cAMP-dependent protein kinase isozymes correlates with the growth inhibitory effect of site-selective 8-Cl-cAMP demonstrated in cancer cell lines (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., Ogreid, D., D?skeland, S.O., Jahnsen, T., and Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6319-6322). Such selective modulation of protein kinase isozyme regulatory subunits was also found in the 8-Cl-cAMP-induced inhibition of both transformation and transforming growth factor alpha (TGF alpha) production in Ki-ras-transformed rat kidney fibroblasts (Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and Cho-Chung, Y. S. (1989) FEBS Lett. 242, 363-367). In this work, we have demonstrated that 8-Cl-cAMP antagonizes the TGF alpha effect in TGF alpha-transformed mouse mammary epithelial cells (NOG-8TFC17) at the level of gene expression for cAMP receptor protein isoforms, RI and RII (the regulatory subunits of protein kinase isozymes). Northern blot analysis demonstrated that in the transformed NOG-8TFC17 cells, compared with the nontransformed counterpart NOG-8 cells, the mRNA levels for the RI alpha cAMP receptor protein markedly increased, whereas the mRNA levels for the RII alpha and RII beta cAMP receptor proteins decreased. 8-Cl-cAMP, which induced growth inhibition and phenotypic reversion in NOG-8TFC17 cells, caused an inverse change in the mRNA patterns of the cAMP receptor proteins; RI alpha cAMP receptor mRNA sharply decreased to levels comparable with that of the nontransformed NOG-8 cells, whereas RII beta mRNA increased to a level even greater than that in the NOG-8 cells. In addition, one mRNA species of RII alpha increased, whereas the other RII alpha mRNA species decreased during the treatment. The mRNA level for the catalytic subunit of protein kinase, however, did not change during 8-Cl-cAMP treatment. In addition, 8-Cl-cAMP brought about a reduction in both TGF alpha mRNA and protein levels. These coordinated changes in the expression of the cAMP receptor proteins and TGF alpha were not observed during cis-hydroxyprolineor TGF beta-induced growth inhibition of the NOG-8TFC17 cells. Thus, the antagonistic effect of 8-Cl-cAMP toward TGF alpha-induced transformation involves modulation of the expression of a specific set of cellular genes.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.     

Alpha 6.beta 1 integrin (laminin receptor) is down-regulated by tumor necrosis factor alpha and interleukin-1 beta in human endothelial cells.

Endothelial cells from human umbilical vein (HEC) express several distinct integrin complexes that mediate the interaction with the basal membrane components. In this paper we show that treatment with tumor necrosis factor alpha (TNF alpha) down-regulates the expression of the laminin receptor alpha 6.beta 1 integrin in cultured HEC. After 48 h of treatment with TNF alpha, the level of expression of the alpha 6.beta 1 complex reached 20% of the control value. The down-regulation of the alpha 6.beta 1 integrin is caused by a decreased expression of the alpha 6 subunit, whereas the synthesis of the beta 1 subunit remains constant. Northern blot analysis shows that the decreased level of alpha 6 subunit synthesis is caused by down-regulation of alpha 6 mRNA in TNF alpha-treated HEC. TNF alpha treatment does not alter the expression of alpha 2, alpha 3, and alpha 5 integrins, also present on endothelial cell surface, thus showing that this cytokine has a selective action on distinct integrin complexes. Down-regulation of alpha 6.beta 1 correlates with pronounced reduction in adhesion of TNF alpha-treated HEC to laminin, but not to fibronectin-coated culture dishes. In addition to TNF alpha, interleukin-1 beta also decreases the expression of the alpha 6.beta 1 integrin and reduces adhesion to laminin, thus suggesting that this regulation plays an important role in inflammation.     

Echinococcus granulosus down regulates the hepatic expression of inflammatory cytokines IL-6 and TNF alpha in BALB/c mice

Hydatid disease is caused by the metacestode of Echinococcus granulosus. Different experimental models have been used to understand hydatid disease. In current studies BALB/c mice were used to evaluate the hepatic response of IL-6 and TNF alpha triggered by Echinococcus granulosus. BALB/c mice were intraperitoneally infected with protoscoleces from E. granulosus; hydatid cysts appeared on the liver eight weeks after inoculation. The RNA extracted from hepatic sections was used for RT-PCR amplification with primers for IL-6, TNF alpha, IL-10, TGF beta and G3PDH. In situ cytokine expression was assessed by FISH. Complete parasite cysts on the liver surface were observed 16 weeks after infection; controls were negative. The expression of IL-6 and TNF alpha was normal at baseline and declined progressively eight weeks after infection; in some animals such expression was abrogated 16 weeks after infection. On the other hand IL-10 and TGF beta were increased progressively. Controls expressed the cytokines normally. Present results suggest that E. granulosus induces a local immunosupression probably mediated by IL-10 and TGF beta; therefore it seems possible that such a mechanism would assist the parasite in escaping the harmful host cell-mediated response.     

Hypothesis: can glucose-insulin-potassium regimen in combination with polyunsaturated fatty acids suppress lupus and other inflammatory conditions?

In systemic lupus erythematosus, plasma concentrations of tumor necrosis factor alpha (TNF alpha) and other pro-inflammatory cytokines are elevated and those of transforming growth factor beta (TGF beta) are decreased. TNF alpha prevents lupus nephropathy whereas increased concentration of TGF beta causes glomerulosclerosis. Insulin inhibits TNF alpha and enhances TGF beta production, augments nitric oxide synthesis and blocks superoxide anion generation. Polyunsaturated fatty acids (PUFAs) also have actions similar to insulin. Hence, it is suggested that a combination of insulin (in the form of glucose-insulin-potassium) and PUFAs may be of benefit in lupus and other inflammatory conditions.