Early response to bleomycin is characterized by different cytokine and cytokine receptor profiles in lungs

The sensitivity to the fibrosis-inducing effect of bleomycin varies considerably from species to species, the reasons for which are unknown. The variability of the response in different strains of mice is well documented. Recent evidence indicates that the upregulated expression of cytokines and cytokine receptors may be involved. We evaluated the expression pattern of some cytokines and their receptors in C57Bl/6J bleomycin-sensitive and Balb/C bleomycin-resistant mice. Animals from both strains received, under ether anesthesia, either saline (50 microl) or bleomycin (0.1 U/50 microl) intratracheally. At various times after the treatment, the lungs were analyzed for cytokines and cytokine receptors by histochemistry and their mRNA by RNase protection assay. A significantly increased expression of TNF-alpha and IL-1beta was observed in both strains. However, an upregulated lung expression for TNF-alpha and IL-1 receptors was observed in C57Bl/6J-sensitive animals only. This profile is evident from 63 h onward. In addition to TNF-alpha, bleomycin administration also resulted in the upregulated expression of TGF-beta in the lungs of both strains at 8 h and in an enhanced expression of TGF-beta receptors I and II in C57Bl/6J mice only. The upregulation of TGF-beta receptor expression was preceded in this strain by an increased expression of IL-4, IL-13, and IL-13 receptor-alpha (at 8 h after bleomycin) and followed by an upregulation of gp130 and IL-6. The difference we observed in the cytokine receptor profile may offer an additional explanation for the different fibrogenic response of the two mouse strains to bleomycin.     

Apoptotic cells and innate immune stimuli combine to regulate macrophage cytokine secretion

Macrophage interactions with apoptotic cells can suppress inflammatory responses, but cell death by apoptosis may also trigger inflammation. We now report that murine macrophages exposed to the combination of apoptotic cells and archetypal ligands for Toll-like receptors (TLRs) 2, 4, and 9 mount cytokine responses that differ importantly from those elicited by either class of stimulus alone. TLR ligands induced early and sustained secretion of TNF-alpha, macrophage-inflammatory protein (MIP) 1alpha and MIP-2 with later secretion of IL-10, IL-12, and TGF-beta1; apoptotic cells alone stimulated late TGF-beta1 secretion only. The combination of apoptotic cells and TLR ligands enhanced early secretion of TNF-alpha, MIP-1alpha, and MIP-2 and increased late TGF-beta1 secretion, while suppressing late TNF-alpha, IL-10, and Il-12 by mechanisms which could nevertheless be overridden by IFN-gamma. We propose that this combinatorial macrophage cytokine response to apoptotic cells and TLR ligands may contribute to recruitment and activation of innate immune defense when cell death occurs at infected inflamed sites while promoting later resolution with diminished engagement of adaptive immunity.     

Modulation of matrix metalloproteinase production from human lung fibroblasts by type 4 phosphodiesterase inhibitors

Over-expression of matrix metalloproteinases by lung fibroblasts has been blamed for much of the tissue destruction associated with airway inflammation. Because cyclic AMP is known to regulate fibroblast proliferation, as well as cytokine and extracellular matrix protein production, the current study was designed to evaluate the ability of three selective phosphodiesterase (PDE) type 4 inhibitors, rolipram, cilomilast and CI-1044, to inhibit extracellular matrix degradation. Using zymography and ELISA, we found that pro-MMP-2 release was enhanced following 24 h treatment of human lung fibroblast (MRC-5) with TGF-beta1 (10 ng/ml) or TNF-alpha (10 ng/ml), whereas PMA (0.02 microM) had no effect. One hour of pre-incubation with PDE4 inhibitors (10 microM) induced an inhibition of TNF-alpha-stimulated pro-MMP-2 release. Zymography and immunoblotting revealed that fibroblasts cultured with PMA or TNF-alpha released increased amounts of pro-MMP-1, whereas TGF-beta1 had no effect. Incubation with CI-1044 or cilomilast significantly prevented the TNF-alpha increase in pro-MMP-1. These results suggest that PDE4 inhibitors are effective in inhibiting the pro-MMP-2 and pro-MMP-1 secretion induced by TNF-alpha and might underline a potential therapeutic benefit of selective PDE4 inhibitors in lung diseases associated with abnormal tissue remodelling.     

Radioprotective efficacy of delta-tocotrienol,a vitamin E isoform,is mediated through granulocyte colony-stimulating factor

Aims

The objectives of this study were to determine the cytokine induction by delta tocotrienol (DT3, a promising radiation countermeasure) and to investigate the role of granulocyte colony-stimulating factor (G-CSF) in its radioprotective efficacy against ionizing radiation in mice.

Main methods

Multiplex Luminex was used to analyze cytokines induced by DT3 and other tocols (gamma-tocotrienol and tocopherol succinate) in CD2F1 mice. Mice were injected with an optimal dose of DT3 and a G-CSF antibody, and their 30-day survival against cobalt-60 gamma-irradiation was monitored. The neutralization of G-CSF by the administration of a G-CSF-specific antibody in DT3-injected mice was investigated by multiplex Luminex.

Key findings

Our data demonstrate that DT3 induced high levels of various cytokines comparable to other tocols being developed as radiation countermeasures. DT3 significantly protected mice against ionizing radiation, and the administration of a G-CSF neutralizing antibody to DT3-treated animals resulted in the complete abrogation of DT3's radioprotective efficacy and neutralization of G-CSF in peripheral blood.

Significance

Our study findings suggest that G-CSF induced by DT3 mediates its radioprotective efficacy against ionizing radiation in mice.     

Inflammatory cytokines augments TGF-beta1-induced epithelial-mesenchymal transition in A549 cells by up-regulating TbetaR-I

Epithelial-mesenchymal transition (EMT) is believed to play an important role in fibrosis and tumor invasion. EMT can be induced in vitro cell culture by various stimuli including growth factors and matrix metalloproteinases. In this study, we report that cytomix (a mixture of IL-1beta, TNF-alpha and IFN-gamma) significantly enhances TGF-beta1-induced EMT in A549 cells as evidenced by acquisition of fibroblast-like cell shape, loss of E-cadherin, and reorganization of F-actin. IL-1beta or TNF-alpha alone can also augment TGF-beta1-induced EMT. However, a combination of IL-1beta and TNF-alpha or the cytomix is more potent to induce EMT. Cytomix, but not individual cytokine of IL-1beta, TNF-alpha or IFN-gamma, significantly up-regulates expression of TGF-beta receptor type I (TbetaR-I). Suppression of TbetaR-I, Smad2 or Smad3 by siRNA partially blocks EMT induction by cytomix plus TGF-beta1, indicating cytomix augments TGF-beta1-induced EMT through enhancing TbetaR-I and Smad signaling. These results indicate that inflammatory cytokines together with TGF-beta1 may play an important role in the development of fibrosis and tumor progress via the mechanism of epithelial-mesenchymal transition.     

Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.     

Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.     

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.     

Mentha piperita (Linn.) leaf extract provides protection against radiation induced chromosomal damage in bone marrow of mice

Oral administration of M. piperita (1 g/kg body weight/day) before exposure to gamma radiation was found to be effective in protecting against the chromosomal damage in bone marrow of Swiss albino mice. Animals exposed to 8 Gy gamma radiation showed chromosomal aberrations in the form of chromatid breaks, chromosome breaks, centric rings, dicentrics, exchanges and acentric fragments. There was a significant increase in the frequency of aberrant cells at 6 hr after irradiation. Maximum aberrant cells were observed at 12 hr post-irradiation autopsy time. Further, the frequency of aberrant cells showed decline at late post-irradiation autopsy time. However, in the animals pretreated with Mentha extract, there was a significant decrease in the frequency of aberrant cells as compared to the irradiated control. Also significant increase in percentage of chromatid breaks, chromosome breaks, centric rings, dicentrics, exchanges, acentric fragments, total aberrations and aberrations/damaged cell was observed at 12 hr post-irradiation autopsy time in control animals, whereas Mentha pretreated irradiated animals showed a significant decrease in percentage of such aberrations. A significant decrease in GSH content and increase in LPO level was observed in control animals, whereas Mentha pretreated irradiated animals exhibited a significant increase in GSH content and decrease in LPO level but the values remained below the normal. The radioprotective effect of Mentha was also demonstrated by determining the LD(50/30) values (DRF = 1.78). The results from the present study suggest that Mentha pretreatment provides protection against radiation induced chromosomal damage in bone marrow of Swiss albino mice.     

Microglia play a role in mediating the effects of cytokines on the structure and function of the rat pineal gland

The role of the pineal gland in regulating immune function has been extensively investigated. However, there is little information about possible feedback mechanisms of immunological factors on pineal gland neuroendocrine functions. Therefore, experiments were designed to test the effects of cytokines (interferon-gamma, IFN-gamma, interleukin-1 beta, IL-1 beta; tumor necrosis factor-alpha, TNF-alpha; transforming growth factor-beta 1, TGF-beta 1) on pinealocytes and the role of pineal microglia in mediating these cytokine effects in the pineal gland of the rat. Our studies showed that IFN-gamma enhanced 5-hydroxytryptamine (5-HT) content (measured by high-performance liquid chromatography, HPLC) and increased pinealocyte process length in pineal cultures. IL-1 beta treatment decreased 5-HT content in both cell and organ culture, but exhibited no effect on pinealocyte process length. 5-HT content and process length were decreased by TNF-alpha treatment. IFN-gamma and IL-1 beta exhibited no significant effect in the absence of microglia in cell cultures. In contrast, TNF-alpha caused a further decline in 5-HT content even in the absence of microglia in the cultures. The effects of TNF-alpha were probably due to toxic effects, since an increased number of pyknotic nuclei were observed in treated cultured explants. TGF-beta 1 treatment caused aggregation of pinealocytes in cultures and suppressed process length and 5-HT content. In conclusion, cytokine effects on pinealocytes may be mediated by microglia (IFN-gamma and IL-1 beta) or act directly on pinealocytes (TNF-alpha). The presence of IL-1 beta and TGF-beta 1 protein in the pineal gland and the suppressive effect of TGF-beta 1 on pinealocytes in cultures further suggest that endogenous cytokines play regulatory roles in response to peripheral homeostatic changes.     

H-2 complex influences cytokine gene expression in Leishmania infantum-infected macrophages

This work aims to study the influence of H-2 locus in the control of Leishmania infantum infection by evaluating whether cytokine responses by host macrophages of different H-2 haplotype are differentially regulated, either induced or actively impaired during parasite growth and replication. This study shows that macrophages of "non-cure" phenotype (H-2(d)) are more susceptible to infection with virulent L. infantum promastigotes. Virulent parasites lead to impaired IL-12 and inhibited TNF-alpha expression. The degree of parasite virulence is an important contributing factor to differences detected in cytokine expression. Virulent parasites also induced TGF-beta, a deactivating cytokine that is known to suppress Th-1 type responses, thus allowing the parasite to subvert antimicrobial activity and increase its chances of survival. Depending on specific host haplotype, cells differentially respond to infection since TNF-alpha expression is inhibited and TGF-beta is enhanced by macrophages of "non-cure" phenotype, thus perhaps determining their degree of susceptibility in this strain of mice.     

Quercetin ameliorates gamma radiation-induced DNA damage and biochemical changes in human peripheral blood lymphocytes

We investigated the radioprotective efficacy of quercetin (QN), a naturally occurring flavonoid against gamma radiation-induced damage in human peripheral blood lymphocytes and plasmid DNA. In plasmid study, QN at different concentrations (3, 6, 12, 24 and 48 microM) were pre-incubated with plasmid DNA for 1h followed by exposure of 6 Gy radiation. Among all concentrations of QN used, 24 microM showed optimum radioprotective potential. To establish the most effective protective concentration of QN in lymphocytes, the cells were pre-incubated with 3, 6, 12, 24 and 48 microM of QN for 30 min and then exposed to 4 Gy gamma-radiation. The concentration-dependent effects of QN were evaluated by scoring micronuclei (MN) frequencies. The results showed that QN decreased the MN frequencies dose dependently, but the effect was more pronounced at 24 microM. Thus, 24 microM of QN was selected as the optimum concentration and was further used to evaluate its radioprotective effect in lymphocytes. For that a separate experiment was carried out, in which lymphocytes were incubated with QN (24 microM) for 30 min and exposed to different doses of radiation (1, 2, 3 and 4 Gy). Genetic damage (MN, dicentric aberration and comet attributes) and biochemical changes were measured to evaluate the effect of QN on gamma-radiations (1-4 Gy). Radiation exposed showed significant increases in the genetic damage and thiobarbituric acid reactive substances (TBARS) accompanied by a significant decrease in the antioxidant status. QN pretreatment significantly decreased the genetic damage and TBARS and improved antioxidant status through its antioxidant potential. Altogether, our findings encourage further mechanistic and in vivo studies to investigate radioprotective efficacy of QN.     

CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.     

Differential induction of TGF-beta regulates proinflammatory cytokine production and determines the outcome of lethal and nonlethal Plasmodium yoelii infections

Transforming growth factor-beta is an essential moderator of malaria-induced inflammation in mice. In this study, we show that the virulence of malaria infections is dependent upon the cellular source of TGF-beta and the timing of its production. C57BL/6 mice infected with a nonlethal (Py17X) strain of Plasmodium yoelii produce TGF-beta from 5 days postinfection; this correlates with resolution of parasitemia, down-regulation of TNF-alpha, and full recovery. In contrast, infection with the lethal strain Py17XL induces high levels of circulating TGF-beta within 24 h; this is associated with delayed and blunted IFN-gamma and TNF-alpha responses, failure to clear parasites, and 100% mortality. Neutralization of early TGF-beta in Py17XL infection leads to a compensatory increase in IL-10 production, while simultaneous neutralization of TGF-beta and IL-10R signaling leads to up-regulation of TNF-alpha and IFN-gamma, prolonged survival in all, and ultimate resolution of infection in 40% of Py17XL-infected animals. TGF-beta production can be induced in an Ag-specific manner from splenocytes of infected mice, and by cross-linking surface CTLA-4. CD25(+) and CD8(+) cells are the primary source of TGF-beta following Py17X stimulation of splenocytes, whereas Py17XL induces significant production of TGF-beta from adherent cells. In mice immunized against Py17XL, the early TGF-beta response is inhibited and is accompanied by significant up-regulation of IFN-gamma and TNF-alpha and rapid resolution of challenge infections.     

Cycling of human dendritic cell effector phenotypes in response to TNF-alpha: modification of the current 'maturation' paradigm and implications for in vivo immunoregulation.

Dendritic cells (DCs) are potent antigen presenting cells reported to undergo irreversible functional 'maturation' in response to inflammatory signals such as TNF-alpha. The current paradigm holds that this DC maturation event is required for full functional capacity and represents terminal differentiation of this cell type, culminating in apoptotic cell death. This provides a possible mechanism for avoiding dysregulated immunostimulatory activity, but imposes constraints on the capacity of DCs to influence subsequent immune responses and to participate in immunological memory. We report that the cell surface and functional effects induced by TNF-alpha are reversible and reinducible. These effects are accompanied by a concordant modulation of cytokine mRNA expression that includes the induction of proinflammatory factors (IL-15, IL-12, LT-alpha, LT-beta, TNF-alpha, RANTES) which is coincident with the down-regulation of counter-regulatory cytokines (IL-10, TGF-beta1, TGF-beta2, IL-1 RA, MCP-1). The resultant net effect is a dendritic cell activation state characterized by a transient proinflammatory posture. These results demonstrate that 1) human DCs do not undergo terminal 'maturation' in response to TNF-alpha, 2) DC phenotypes are more pleiotropic than previously thought, and 3) DCs are potential immunoregulatory effector cells with implications for control of immune responses in both in vivo and in vitro systems.