Recombinant TNF-alpha mediated regulation of the I kappa B-alpha/NF-kappa B signaling pathway: evidence for the enhancement of pro- and anti-inflammatory cytokines in alveolar epithelial cells

The signaling transduction mechanism mediated by tumor necrosis factor-alpha (TNF-alpha) in the alveolar epithelium is not well characterized. It was subsequently hypothesized that recombinant murine TNF-alpha (rmTNF-alpha) selectively regulates the inhibitory kappa B (I kappa B-alpha)/nuclear factor-kappa B (NF-kappa B) pathway and interferes with the endogenous biosynthesis of pro-inflammatory (stimulatory) and anti-inflammatory (inhibitory) cytokines. The cytokine rmTNF-alpha induced, in a time- and dose-dependent manner, the degradation of I kappa B-alpha within the cytosolic compartment, an effect associated with up-regulating its phosphorylation. This allowed the biphasic regulation of selective NF-kappa B subunit nuclear translocation, thereby mediating a dual excitatory mechanism on NF-kappa B activation. The immunoregulatory effect of rmTNF-alpha was associated with a time-dependent induction of pro-inflammatory [interleukin (IL)-1 beta, IL-6 and TNF-alpha] and anti-inflammatory (IL-10) cytokine biosynthesis. These results indicate a novel involvement of an I kappa B-alpha/NF-kappa B-sensitive pathway mediating the effect of TNF-alpha, which is associated with an autocrine, endogenous mechanism mediating the regulation of cytokine signaling.     

Effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines.

We evaluated the effects of binary combinations of four cytokines on production of the positive acute phase proteins alpha-1 antichymotrypsin, haptoglobin and fibrinogen, and the negative acute phase proteins albumin and alpha-fetoprotein (AFP) in two human hepatoma cell lines. The effects of the cytokine combinations on the five proteins varied; each protein exhibited a unique and specific pattern of response to the cytokine combinations. In Hep G2 cells, antichymotrypsin was induced by all four cytokines, IL-6, IL-1, TNF-alpha, and transforming growth factor beta 1 alone, and their effects in binary combinations could be attributed to additive or minimally synergistic interactions. Fibrinogen was induced only by IL-6 and this induction was inhibited by IL-1 alpha, TNF-alpha or transforming growth factor beta 1. Haptoglobin was also induced only by IL-6, but TNF-alpha was the only cytokine that inhibited this induction at all concentrations of IL-6. Each of the four cytokines alone down regulated production of AFP and albumin. However, binary combinations of the four cytokines were simply additive, for the most part, in inhibiting AFP production, whereas the inhibitory effects of combinations of cytokines on albumin production differed significantly from simple additive effects. These observations, taken together with studies of effects of cytokine combinations on other acute phase proteins, indicate that the various acute phase proteins respond differently to different combinations of cytokines and that the potential exists for highly specific regulation of synthesis of individual plasma proteins by cytokine interactions. These findings imply that the acute phase response in vivo represents the integrated sum of multiple, separately regulated changes in gene expression.     

Lactoferrin: influences on Langerhans cells, epidermal cytokines, and cutaneous inflammation.

It has been suggested previously that, in addition to other biological roles, lactoferrin (LF) may display antiinflammatory properties secondary to the regulation of cytokine expression. To explore this concept further, we have here examined in human volunteers the influence of recombinant homologous LF on the migration of epidermal Langerhans cells (LC), a process that is known to be dependent upon the local availability of certain proinflammatory cytokines including tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta). In common with previous studies in mice, it was found that topical administration of LF prior to exposure at the same site to the contact sensitizer diphenylcyclopropenone resulted in a significant reduction of allergen-induced LC migration from the epidermis (measured as a function of the frequency of CD1a+ or HLA-DR+ LC found in epidermal sheets prepared from punch biopsies of the treated skin sites). However, under the same conditions of exposure, LF was unable to influence migration of LC induced by the intradermal administration of TNF-alpha data consistent with the hypothesis that one action of LF in the skin is to regulate the local production of this cytokine. Further support for this hypothesis was derived from experiments conducted with IL-1beta. This cytokine is also able to induce the mobilization of LC following intradermal injection, although in this case, migration is known to be dependent upon the de novo production of TNF-alpha. We observed that prior exposure to LF resulted in a substantial inhibition of IL-1beta-induced LC migration, data again consistent with the regulation of TNF-alpha production by LF. Collectively, these results support the view that LF is able to influence cutaneous immune and inflammatory processes secondary to regulation of the production of TNF-alpha and possibly other cytokines.     

TNF-alpha, soluble TNF receptor and interleukin-6 plasma levels in the general population

The cytokines tumor necrosis factor-alpha (TNF-alpha), soluble TNF receptors p55 and p75, and interleukin 6 (IL-6) are involved in host defense against several microbiological agents, in the process of inflammation and also in body weight regulation. In the present study, we sought to assess the influence of age, gender, smoking, and body mass index on plasma levels of TNF-alpha, TNF receptors, and IL-6 in more than 550 adult subjects randomly selected from the Bavarian population. None of the cytokine parameters had a normal distribution and all distributions were significantly skewed. The cytokine plasma levels investigated increased significantly with age, while gender had a relatively weaker influence on the plasma levels. Plasma levels of TNF-alpha, TNF receptors, and IL-6 correlated significantly with the BMI. The study provides insights into factors influencing the cytokine levels investigated in a randomly chosen study sample.     

Activation of nuclear factor kappaB and induction of apoptosis by tumor necrosis factor-alpha in the mouse uterine epithelial WEG-1 cell line

In order to better understand how tumor necrosis factor (TNF)-alpha may contribute to the local regulation of uterine cell death, cultures of mouse uterine epithelial WEG-1 cells were exposed to TNF-alpha and observed at different time intervals. Earliest decrease in cell viability was observed after 31 h of exposure to 50 ng/ml mouse TNF-alpha and was associated with the expression of several markers of apoptosis. Treatment with human TNF-alpha or addition of a neutralizing antibody against TNF-alpha receptor protein 80 to mouse TNF-alpha resulted in attenuated induction of apoptosis, suggesting that coengagement of the two TNF-alpha receptor types is required for maximal impact. Ceramide analogs failed to replicate the effect of TNF-alpha and the stress-activated protein kinase signaling pathway was not activated by the cytokine. Treatment with mouse TNF-alpha resulted in an increase in nuclear factor (NF)kappaB activity that receded after 24 h. The impact of human TNF-alpha on NFkappaB activation was more moderate. Addition of either one of three different inhibitors of NFkappaB (SN50, PDTC, and A771726) to mouse TNF-alpha sensitized WEG-1 cells to the toxicity of the cytokine. Our data suggest that WEG-1 cells initiate their response to TNF-alpha with an increase in NFkappaB activation that may have transiently biased these cells toward cell death resistance.     

The intestinal microflora regulates cytokine production positively in spleen-derived macrophages but negatively in bone marrow-derived macrophages.

Besides its role as a barrier against potential pathogens, intestinal flora is presumed to protect the host by priming the immunological defense mechanisms. In this respect, the influence of intestinal flora on macrophage precursors was examined, and its modulating effect was compared on LPS-induced cytokine production by macrophages derived from bone marrow and spleen precursors (BMDM and SDM respectively). The regulation of IL-1, IL-6, TNF-alpha and IL-12 production in macrophages from germ-free and from three groups of flora-associated mice, conventional, conventionalized and E. coli-mono-associated mice, was investigated. The whole flora inhibited IL-1, TNF-alpha and IL-12 secretion by BMDM, whereas it had a stimulatory effect on IL-12 secretion by SDM. Implantation of E. coli alone enhanced cytokine secretion by BMDM but had a more limited effect than whole flora on SDM, enhancing only TNF-alpha and IL-12 secretion. Study of expression of mRNA showed a correlation with protein secretion for IL-6 but not for TNF-alpha and IL-1. IL-12 enhancement in BMDM seemed to be dependent on regulation of p35 mRNA expression while it was correlated to increased p40 mRNA expression in SDM. The results demonstrated that intestinal flora modulated bone marrow and spleen macrophage cytokine production in a differential manner and suggested a role for bacteria other than E. coli among the whole flora. The contrasting effects exerted by the intestinal flora on bone marrow and spleen precursors are an interesting observation in view of the different functions of these organs in immunity. The finding that intestinal flora enhanced IL-12 production in spleen is also potentially important since this cytokine is implicated in the determination of the relative levels of Th1 and Th2 responses and plays a pivotal role in host defense against intracellular microorganisms.     

Aldose reductase regulates TNF-alpha-induced cell signaling and apoptosis in vascular endothelial cells

TNF-alpha, generated during the systemic inflammatory response, triggers a wide range of biological activities that mediate the neurologic manifestations associated with cancer and infection. Since this cytokine regulates ion channels in vitro (especially Kv1.3 and Kir2.1), we aimed to study Kv1.3 and Kir2.1 expression in brain in response to in vivo systemic inflammation. Cancer-induced cachexia and LPS administration increased plasma TNF-alpha. Kv1.3 and Kir2.1 expression was impaired in brain during cancer cachexia. However, LPS treatment induced Kv1.3 and downregulated Kir2.1 expression, and TNF-alpha administration mimicked these results. Experiments using TNF-alpha double receptor knockout mice demonstrated that the systemic inflammatory response mediates K(+) channel regulation in brain via TNF-alpha-dependent and -independent redundant pathways. In summary, distinct neurological alterations associated with systemic inflammation may result from the interaction of various cytokine pathways tuning ion channel expression in response to neurophysiological and neuroimmunological processes.     

Tumor necrosis factor-alpha/interleukin-10 balance in normal and cystic fibrosis children

BACKGROUND: The balance between tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) is important for immune homeostasis maintenance. Exuberant production of TNF-alpha contributes to overwhelming inflammatory response and tissue damage. But, commonly, increase in TNF-alpha is counterbalanced by simultaneous synthesis of an anti-inflammatory cytokine IL-10, which suppresses production of many activating and regulatory mediators. AIMS: In the present study, the relationships between TNF-alpha and IL-10 in the plasma of healthy school-children and cystic fibrosis (CF) patients have been investigated. METHODS: Blood samples were obtained from 12 CF patients with chronic pulmonary disease and 18 healthy schoolchildren vaccinated with live attenuated rubella vaccine. IL-10 and TNF-alpha were determined in the plasma samples using commercially available enzyme-linked immunosorbent assay kits. RESULTS: Before vaccination, most healthy children (13 of 18) demonstrated superiority of pro-inflammatory TNF-alpha over anti-inflammatory IL-10 (TNF-alpha/IL-10 > 1). In these subjects, a significant positive linear association between the cytokine values has been found. Vaccine challenge resulted in a marked reduction of TNF-alpha/IL-10 ratios. In addition, a disappearance of correlation between the cytokine values was observed. Such disturbance was related to exuberant elevation of the IL-10 levels after inoculation. On the contrary, in CF individuals, plasma cytokine values remained in strong linear association independently of TNF-alpha or IL-10 predominance. No spikes in the plasma levels of IL-10 in CF patients during a 6-month observation period have been revealed. CONCLUSIONS: There were no fundamental differences between CF and healthy children in the regulation of TNF-alpha and IL-10 secretion. Thus, immune quiescence seemed to be associated with the predominance of TNF-alpha, whereas immune disturbance was characterized by IL-10 superiority. The only abnormality that was found in CF patients consisted of their inability to produce unlimitedly IL-10 in response to antigen stimuli.     

Paradoxical anti-inflammatory actions of TNF-alpha: inhibition of IL-12 and IL-23 via TNF receptor 1 in macrophages and dendritic cells

IL-12 and TNF-alpha are central proinflammatory cytokines produced by macrophages and dendritic cells. Disregulation of TNF-alpha is associated with sepsis and autoimmune diseases such as rheumatoid arthritis. However, new evidence suggests an anti-inflammatory role for TNF-alpha. TNF-alpha-treated murine macrophages produced less IL-12p70 and IL-23, after stimulation with IFN-gamma and LPS. Frequency of IL-12p40-producing macrophages correspondingly decreased as measured by intracellular cytokine staining. IL-12p40 production was also inhibited in dendritic cells. TNFR1 was established as the main receptor involved in IL-12p40 regulation, because IL-12p40 levels were not affected by TNF-alpha in TNFR1(-/-)-derived macrophages. Macrophages activated during Listeria monocytogenes infection were more susceptible to inhibition by TNF-alpha than cells from naive animals, which suggests a regulatory role for TNF-alpha in later stages of infection. This nonapoptotic anti-inflammatory regulation of IL-12 and IL-23 is an important addition to the multitude of TNF-alpha-induced responses determined by cell-specific receptor signaling.     

Tumor necrosis factor-alpha-induced insulin resistance in adipocytes

Recent studies examining the link between insulin resistance and the development of obesity and noninsulin-dependent diabetes mellitus are consistent with the involvement of tumor necrosis factor-alpha (TNF-alpha) as a central mediator. In insulin resistant obese mouse models, neutralization of TNF-alpha in circulation has been demonstrated to restore insulin-mediated glucose uptake. Adipose tissue has been shown to be a site for synthesis of TNF-alpha, with the degree of adiposity directly correlated with the level of synthesis. Studies conducted on obese human patients have demonstrated a correlation between levels of TNF-alpha, the extent of obesity, as well as the level of hyperinsulinemia observed. Mechanistic studies in cell culture have suggested that TNF-alpha functions to render cells insulin resistant through regulation of the synthesis of the insulin responsive glucose transporter as well as through interference with insulin signaling. This review will address these issues and additionally introduce the reader to the molecular aspects of TNF-alpha, its receptors as well as TNF-alpha-initiated signaling cascades, that are necessary to understand the function of this cytokine in the regulation of adipose tissue metabolism.     

Synergistic induction of nuclear factor-kappaB by transforming growth factor-beta and tumour necrosis factor-alpha is mediated by protein kinase A-dependent RelA acetylation

The TGF-beta (transforming growth factor-beta) pathway represents an important signalling pathway involved in regulating diverse biological processes, including cell proliferation, differentiation and inflammation. Despite the critical role for TGF-beta in inflammatory responses, its role in regulating NF-kappaB (nuclear factor-kappaB)-dependent inflammatory responses still remains unknown. In the present study we show that TGF-beta1 synergizes with proinflammatory cytokine TNF-alpha (tumour necrosis factor-alpha) to induce NF-kappaB activation and the resultant inflammatory response in vitro and in vivo. TGF-beta1 synergistically enhances TNF-alpha-induced NF-kappaB DNA binding activity via induction of RelA acetylation. Moreover, synergistic enhancement of TNF-alpha-induced RelA acetylation and DNA-binding activity by TGF-beta1 is mediated by PKA (protein kinase A). Thus the present study reveals a novel role for TGF-beta in inflammatory responses and provides new insight into the regulation of NF-kappaB by TGF-beta signalling.     

A bacteria-induced switch of sympathetic effector mechanisms augments local inhibition of TNF-alpha and IL-6 secretion in the spleen.

It is believed that an inflammation-induced activation of the CNS leads to an inhibition of overshooting immune responses to prevent extensive local cytokine secretion. However, immunosuppression by the sympathetic nervous system may be unfavorable when bacteria are present locally and when TNF-alpha is necessary to overcome infection. We now report in a superfusion model, using mouse spleen slices, that although local Pseudomonas aeruginosa increased splenic TNF-alpha and IL-6 secretion severalfold over basal levels, electrically released neurotransmitters attenuated cytokine secretion to similar basal level as under bacteria-free conditions. Bacteria reversed noradrenergic inhibitory effector mechanisms: Under bacteria-free conditions, TNF-alpha secretion was very low and IL-6 secretion was mainly inhibited by alpha2-adrenoreceptor ligation. In the presence of bacteria, TNF-alpha and IL-6 secretion were high and IL-6 secretion was mainly inhibited by beta-adrenoreceptor ligation. The alpha- to beta-adrenoswitch of IL-6 inhibition in the presence of bacteria was mediated by the prior adrenergic regulation of TNF-alpha. In vivo, chemical abrogation of sympathetic inhibition reduced accumulation of bacteria in the spleen, which depended at least in part on TNF-alpha. This suggests that activation of the sympathetic nervous system may be a forerunner for accumulation of bacteria in tissue and consecutively sepsis due to intensified inhibition of TNF-alpha secretion.     

Impact of VIP and cAMP on the regulation of TNF-alpha and IL-10 production: implications for rheumatoid arthritis

Vasoactive intestinal peptide (VIP) is an anti-inflammatory immunomodulatory neuropeptide with therapeutic potential demonstrated for collagen-induced arthritis. The aim of this study was to characterise its potential anti-arthritic effect on human monocytes, macrophages, T cells, and rheumatoid arthritis synovial membrane cells. Monocytes, macrophages, and T cells derived from human peripheral blood were treated with VIP and compared with other cAMP-elevating drugs for a range of activating stimuli. Cytokine production was assessed for cell cultures and, in addition, the ability of VIPs to activate cAMP response element binding protein. VIP partially suppressed monocyte- and macrophage-derived tumour necrosis factor alpha (TNF-alpha) with no effect on IL-10, whereas VIP fails to regulate IL-10 and TNF-alpha production by T lymphocytes. No such modulation of cytokine profile was observed for rheumatoid arthritis synovial membrane cells. Elevation of intracellular cAMP, on the other hand, potently suppressed macrophage TNF-alpha production and modulated T-cell response by inhibiting TNF-alpha and IFN-gamma. VIP's lack of effect on IL-10 and its slight effect on TNF-alpha results from cAMP being rapidly degraded as the phosphodiesterase IV inhibitor, rolipram, rescues cAMP-dependent activation of cAMP response element binding protein. Interestingly, macrophages stimulated with phorbol 12-myristate 13-acetate/ionomycin displayed an augmented IL-10 response upon addition of dibutyryl cAMP, with corresponding downregulation in TNF-alpha, suggesting a complex interaction between protein kinase C and protein kinase A in cytokine regulation. In conclusion, VIP may represent an efficaceous anti-arthritic treatment modulating macrophage and T-cell cytokine profiles when used alongside a phosphodiesterase inhibitor.