Sex steroid-mediated regulation of macrophage/monocyte function in a two-hit model of trauma-hemorrhage and sepsis

Studies have shown that 17beta-estradiol has salutary effects on immune functions after trauma-hemorrhage (TH). It remains unknown, however, whether 17beta-estradiol has a similar effect in a double-hit model of TH and subsequent sepsis. It is also unknown if under those conditions the circulating immune cells accurately represent immunological responses occurring in fixed tissues, such as the spleen. To study this, pre-castrated mice were hormonally treated and then subjected to soft-tissue trauma (i.e. midline laporatomy), hemorrhagic shock (MAP 35+/-5mmHg for 90 min followed by resuscitation) and 24 h later sepsis was induced by cecal ligation and puncture (CLP). Splenic macrophages (SMphi) and peripheral blood mononuclear cells (PBMC) were isolated and cultured with LPS. 5alpha-Dihydrotestosterone-treated mice showed a depressed pro-inflammatory cytokine production after TH-sepsis in both SMphi and PBMC. In contrast, the 17beta-estradiol treated groups showed suppressed pro-inflammatory cytokine production in the PBMC population under those conditions. In summary, 17beta-estradiol was able to prevent immune dysfunction after TH and subsequent sepsis. However, the beneficial effects of 17beta-estradiol were limited to tissue-fixed Mphi, suggesting compartmentalization of the response. Thus, events occurring in the tissue-fixed cells are not necessarily reflected in the circulating PBMC population.     

Tissue compartment-specific role of estrogen receptor subtypes in immune cell cytokine production following trauma-hemorrhage.

Although 17beta-estradiol administration following trauma-hemorrhage attenuates plasma cytokines and alteration in immune cell cytokine production, it is not known whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta. Accordingly, we examined which ER subtype predominantly mediates the salutary effects of 17beta-estradiol on systemic inflammatory response/immune cell cytokine production in various tissues following trauma-hemorrhage. Male rats underwent trauma-hemorrhage (mean blood pressure: 40 mmHg for 90 min) and fluid resuscitation. The ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), the ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), 17beta-estradiol (50 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation, and various measurements were made 24 h thereafter. 17beta-Estradiol or PPT administration following trauma-hemorrhage prevented the increase in plasma IL-6 and IL-10 levels that were observed in vehicle-treated animals. IL-6 and TNF-alpha production by Kupffer cells increased; however, splenic macrophages (SMPhi), alveolar macrophages (AMPhi), and peripheral blood mononuclear cells (PBMC) had decreased release of these cytokines after trauma-hemorrhage. IL-10 production, however, increased in all macrophage populations. Administration of 17beta-estradiol following trauma-hemorrhage prevented all of these alterations. PPT had the same effects as 17beta-estradiol on IL-6 and TNF-alpha production by Kupffer cells and SMPhi, and DPN had the same effects on AMPhi and PBMC. The same effects as 17beta-estradiol on IL-10 production were observed by PPT on Kupffer cells and DPN on PBMC. Both agonists were equally effective on SMPhi and AMPhi. Thus ER subtypes have tissue compartment-specific roles in mediating the effects of 17beta-estradiol on immune cell functions following trauma-hemorrhage.     

Androstenediol administration after trauma-hemorrhage attenuates inflammatory response, reduces organ damage, and improves survival following sepsis

Although androstenediol (adiol or 5-androstene-3beta,17beta-diol), a metabolite of dehydroepiandrosterone (DHEA), has protective effects following trauma-hemorrhage (T-H), it remains unknown whether administration of adiol has any salutary effects on the inflammatory response and outcome following a combined insult of T-H and sepsis. Male rats underwent T-H shock [mean arterial pressure (MAP) 40 mmHg for 90 min] followed by resuscitation. Adiol (1 mg/kg body wt) or vehicle was administered at the end of resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) at 20 h after T-H or sham operation. Five hours after CLP, plasma and tissue samples were analyzed for cytokines (IL-6 and IL-10), MPO, neutrophil chemotactic factor (CINC-3), and liver injury (alanine aminotransferase and lactate dehydrogenase). In another group of rats, the gangrenous cecum was removed at 10 h after CLP, the cavity was irrigated with warm saline and closed in layers, and mortality was recorded over 10 days. T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil cell activation, and resulted in liver injury. Adiol administration prevented the increase in cytokine production, neutrophil cell activation, and attenuated liver injury. Moreover, rats subjected to the combined insult, receiving vehicle or adiol, had a 50% and 6% mortality, respectively. Since adiol administration suppresses proinflammatory cytokines, reduces liver damage, and decreases mortality after the combined insult of T-H and sepsis, this agent appears to be a novel adjunct to fluid resuscitation for decreasing T-H-induced septic complications and mortality.     

Protective effects of finasteride on the pulmonary immune response in a combined model of trauma-hemorrhage and polymicrobial sepsis in mice

Literature supports findings about a gender specific outcome following multiple trauma. Male sex hormones such as dihydrotestosterone (DHT) exert deleterious effects on the posttraumatic immune response whereas increased estradiol concentrations are correlated with improved outcome. Pretreatment with the 5α-reductase inhibitor finasteride resulted in an improved outcome following trauma-hemorrhage (TH) in mice. The present study tested the hypothesis that finasteride exerts beneficial effects on the posttraumatic immune response also in a combined setting of TH and sepsis when administered during the resuscitation process.

Material and Methods

Male C57BL/6N-mice were subjected to TH (blood pressure, 35 mm Hg, 60 min) followed by finasteride application and fluid resuscitation. Thereafter, finasteride was administered every 12 h. 24 h after TH, sepsis was induced by cecal ligation and puncture (CLP) or sham operation was performed. Plasma cytokines (MIP-1α, MIP-1β, TNF-α, MCP-1, IL-6), productive capacity by alveolar macrophages (AM) and systemic estradiol levels were determined 4 h thereafter. The expression of pro-inflammatory mediators in lung tissue was evaluated by PCR. Pulmonary infiltration of PMN was determined by immunohistochemical staining.

Results

Finasteride treatment resulted in a reduced posttraumatic cytokine secretion of AM as well as in a decreased concentration of MCP-1 and MIP-1β in lung tissue. Systemic estradiol levels were increased following finasteride treatment.

Conclusion

Finasteride mediates salutary effects on the pulmonary immune response using a therapeutical approach following TH–CLP in mice. Thus, finasteride might represent a relevant therapeutic substance following major trauma also in the clinical setting.     

Swertianolin ameliorates immune dysfunction in sepsis via blocking the immunosuppressive function of myeloid-derived suppressor cells

In this study, we studied the long-term proliferation trajectory of myeloid-derived suppressor cells (MDSCs) in murine sepsis model and investigated whether swertianolin could modulate the immunosuppressive function of MDSCs. A murine sepsis model was established by cecal ligation and perforation (CLP), according to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. The bone marrow and spleen of the mice were collected at 24 h, 72 h, 7 and 15 d after sepsis induction. The proportions of monocytic- MDSCs (M-MDSCs; CD11b⁺LY6G^–LY6C^hi) and granulocytic-MDSCs (G-MDSC, CD11b⁺ Ly6G⁺ Ly6C^low) were analyzed by flow cytometry. Then, we have investigated whether swertianolin could modulate the immunosuppressive function of MDSCs in in vitro experiments. G-MDSCs and M-MDSCs increased acutely after sepsis with high levels sustained over a long period of time. G-MDSCs were the main subtype identified in the murine model of sepsis with polymicrobial peritonitis. Furthermore, it was found that swertianolin reduced significantly interleukin-10 (IL-10), nitric oxide (NO), reactive oxygen species (ROS), and arginase production in MDSCs, while reducing MDSC proliferation and promoting MDSC differentiation into dendritic cells. Swertianolin also improved T-cell activity by blocking the immunosuppressive effect of MDSCs. Both subsets of MDSCs significantly increased in the bone marrow and spleen of the mice with sepsis, with GMDSCs being the main subtype identified. Swertianolin effectively regulated the functions of MDSCs and reduced immune suppression.Key words: Sepsis, myeloid-derived suppressor cells (MDSCs), immunosuppression, swertianolin     

Role of IL-10 in regulating proinflammatory cytokine release by Kupffer cells following trauma-hemorrhage

IL-6 and TNF-alpha production by Kupffer cells is markedly stimulated following trauma-hemorrhage (T-H). Because IL-10 is an anti-inflammatory cytokine, the aim of this study was to determine whether IL-10 regulates Kupffer cell proinflammatory cytokine release following T-H. To study this, we subjected adult male Sprague-Dawley rats to sham operation or T-H. The procedure involved a 5-cm midline laparotomy and approximately 90 min of hemorrhagic shock (35 mmHg), followed by resuscitation with four times the shed blood volume in the form of Ringer's lactate. At 2 h after the end of resuscitation, livers were perfused in vitro and perfusate was collected. In separate studies, Kupffer cells were isolated and incubated with different concentrations of anti-IL-10 MAb. IgG was used as control. After 16 h of incubation, IL-6 and TNF-alpha levels were measured by ELISA. Plasma IL-10 levels increased significantly following T-H. IL-10 levels in the perfusate and IL-10 production by cultured Kupffer cells were also significantly higher in the T-H group. When Kupffer cells were incubated with 10 microg/ml of anti-IL-10 MAb, IL-6 and TNF-alpha production were significantly increased in both sham and T-H groups compared with those not treated with anti-IL-10 MAb. However, these changes were not observed when the cells were incubated with irrelevant (control) IgG. These results indicate that IL-10 production by Kupffer cells early after T-H may play a pivotal role in attenuating the proinflammatory cytokine environment, possibly in an autocrine/paracrine manner.     

Impact of sex and age on bone marrow immune responses in a murine model of trauma-hemorrhage.

Although studies have demonstrated that trauma markedly alters the bone marrow immune responses, sex and age are crucial determinants under such conditions and have not been extensively examined. To study this, 21- to 27-day-old (premature), 6- to 8-wk-old (mature), and 20- to 24-mo-old (aged) male and female (proestrus) C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30 +/- 5 mmHg for 90 min) followed by fluid resuscitation. Twenty-four hours after resuscitation, bone marrow cells were harvested. Trauma-hemorrhage induced an increased number of the early pluripotent stem cell-associated bone marrow cell subsets (Sca1(+)CD34(-)CD117(+/-)lin(+/-)) in young mice. The CD117(+) proportion of these cell subsets increased in mature proestrus females, but not in males. Aged males displayed significant lower numbers of Sca1(+)CD34(-)CD117(+/-)lin(+/-) cells compared with young male mice. Trauma-hemorrhage also increased development of granulocyte/macrophage progenitor cells (CD11b(+)Gr-1(+)). Proliferative responses to granulocyte macrophage colony-stimulating factor were maintained in mature and aged proestrus females, but decreased in young mice and mature males. Augmented differentiation into monocyte/macrophage lineage in mature and aged proestrus females was observed and associated with the maintained release of TNF-alpha and IL-6. Conversely, increased IL-10 and PGE(2) production was observed in the male trauma-hemorrhage groups. Thus, sex- and age-specific effects in bone marrow differentiation and immune responses after trauma-hemorrhage occur, which are likely to contribute to the sex- and age-related differences in the systemic immune responses under such conditions.     

Forced expression of stabilized c-Fos in dendritic cells reduces cytokine production and immune responses in vivo

Lim kinase 2 isoforms, LIMK2a and LIMK2b, phosphorylate cofilin leading to remodeling of actin cytoskeleton during neuronal differentiation. The expression and function of the LIMK2d isoform, missing the kinase domain, remain unknown. We analyzed the expression of LIMK2 splice variants in adult rat brain and in cultures of rat neural stem cells by RT-QPCR. All three splice variants were expressed in adult cortex, hippocampus and cerebellum. Limk2a and Limk2d expression, but not Limk2b, increased during neuronal differentiation. We studied the localization and function of LIMK2d isoform by transfecting Hela, NSC-34, and hippocampal rat neuron cultures. Similarly to LIMK2b, LIMK2d was expressed in the cytoplasm, neurites and dendritic spines, but not in the nucleus. Similarly to LIMK2a, LIMK2d over-expression in NSC-34 cells increased neurite length, but independently of cofilin phosphorylation or of direct interaction with actin. Overall, these results indicate that LIMK2d is a third LIMK2 isoform which regulates neurite extension and highlights the possible existence of a kinase independent function of LIMK2.     

Sustained-release prostacyclin analog ONO-1301 ameliorates tubulointerstitial alterations in a mouse obstructive nephropathy model

Tubulointerstitial injuries are crucial histological alterations that predict the deterioration of renal function in chronic kidney disease. ONO-1301, a novel sustained-release prostacyclin analog, accompanied by thromboxane synthase activity, exerts therapeutic effects on experimental pulmonary hypertension, lung fibrosis, cardiomyopathy, and myocardial ischemia, partly associated with the induction of hepatocyte growth factor (HGF). In the present study, we examined the therapeutic efficacies of ONO-1301 on tubulointerstitial alterations induced by unilateral ureteral obstruction (UUO). After inducing unilateral ureteral obstruction in C57/BL6J mice, a single injection of sustained-release ONO-1301 polymerized with poly (D,L-lactic-co-glycolic acid) sustained-release ONO-1301 (SR-ONO) significantly suppressed interstitial fibrosis, accumulation of types I and III collagen, increase in the number of interstitial fibroblast-specific protein-1 (FSP-1)(+) cells, and interstitial infiltration of monocytes/macrophages (F4/80(+)) in the obstructed kidneys (OBK; day 7). Treatment with SR-ONO significantly suppressed the increase of the renal levels of profibrotic factor TGF-β and phosphorylation of Smad2/3, and elevated the renal levels of HGF in the OBK. In cultured mouse proximal tubular epithelial cells (mProx24), ONO-1301 significantly ameliorated the expression of fibroblast-specific protein-1 and α-smooth muscle actin as well as phosphorylation of Smad3 and increased the expression of zonula occludens-1 and E-cadherin in the presence of TGF-β1 as detected by immunoblot and immunocytochemistry, partly dependent on PGI(2) receptor-mediated signaling. Administration of rabbit anti-HGF antibodies, but not the control IgG, partly reversed the suppressive effects of SR-ONO on tubulointerstitial injuries in the OBK. Taken together, our findings suggest the potential therapeutic efficacies of ONO-1301 in suppressing tubulointerstitial alterations partly mediated via inducing HGF, an antifibrotic factor counteracting TGF-β.     

Local regulation of immune responses: corneal endothelial cells alter t cell activation and cytokine production

Corneal endothelial cells (CE cells) inhibit antigen- and mitogen-activated lymphocyte proliferation assays, although interleukin 2 receptor (IL-2R) expression and responsiveness to exogenous IL-2 are unaffected. To examine this activity further, co-cultures of CE cells and T cell clones were studied. CE cells inhibited IL-2 and IL-4 production by T cells stimulated with Ag and APC, but not IL-5 or IL-6 production. CE cells also inhibited NFAT-driven lacZ reporter gene production following Ag stimulation of transfected KZO T hybridoma cells. Conversely, stimulation of IL-2 production by ionomycin, with or without PMA, was unaffected by the CE cells. Preincubation of KZO hybridoma or Jurkat cells with CE cells, or CE cell-conditioned culture supernatant, inhibited the intracellular calcium ([Ca(2+)](i)) increase induced by TCR ligation, but not the [Ca(2+)](i)increase induced by ionomycin or thapsigargin. The inhibitory effect was independent of APC and did not act by blocking costimulation, since IL-2 production stimulated by immobilized anti-CD3 alone was also inhibited by CE cells. The supernatant factor was heat labile. This novel activity is unlike other immunoregulatory molecules, including transforming growth factor beta (TGF-beta) and may contribute to local immune privilege.     

OxLDL immune complexes activate complement and induce cytokine production by MonoMac 6 cells and human macrophages

Oxidized low density lipoprotein (OxLDL) is immunogenic and induces autoimmune responses in humans. OxLDL antibodies are predominantly of the proinflammatory IgG1 and IgG3 isotypes. We tested the capacity of immune complexes prepared with copper-oxidized human LDL and affinity chromatography-purified human OxLDL antibodies [OxLDL-immune complexes (ICs)] to activate complement and to induce cytokine release by MonoMac 6 (MM6) cells and by primary human macrophages. The levels of C4d and C3a were significantly higher in human serum incubated with OxLDL-ICs than after incubation with OxLDL or OxLDL antibody, indicating complement activation by the classical pathway. MM6 cells and primary human macrophages were incubated with OxLDL-ICs, with or without prior conditioning with interferon-gamma. After 18 h of incubation, both MM6 cells and primary human macrophages released significantly higher levels of proinflammatory cytokines after incubation with OxLDL-ICs than after incubation with OxLDL or with OxLDL antibody, both in primed and unprimed cells. OxLDL-ICs were more potent activators of MM6 cells than keyhole limpet hemocyanin-ICs. Blocking Fc gamma receptor I (FcgammaRI) with monomeric IgG1 significantly depressed the response of MM6 cells to OxLDL-ICs. In conclusion, human OxLDL-ICs have proinflammatory properties, as reflected by their capacity to activate the classical pathway of complement and to induce proinflammatory cytokine release from MM6 cells and primary human macrophages.     

Salutary effects of 17beta-estradiol on T-cell signaling and cytokine production after trauma-hemorrhage are mediated primarily via estrogen receptor-alpha

Although 17-estradiol (E2) administration following trauma-hemorrhage prevents the suppression in splenocyte cytokine production, it remains unknown whether the salutary effects of 17-estradiol are mediated via estrogen receptor (ER)- or ER-. Moreover, it is unknown which signaling pathways are involved in 17-estradiol's salutary effects. Utilizing an ER-- or ER--specific agonist, we examined the role of ER- and ER- in E2-mediated restoration of T-cell cytokine production following trauma-hemorrhage. Moreover, since MAPK, NF-B, and activator protein (AP)-1 are known to regulate T-cell cytokine production, we also examined the activation of MAPK, NF-B, and AP-1. Male rats underwent trauma-hemorrhage (mean arterial pressure 40 mmHg for 90 min) and fluid resuscitation. ER- agonist propyl pyrazole triol (PPT; 5 µg/kg), ER- agonist diarylpropionitrile (DPN; 5 µg/kg), 17-estradiol (50 µg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation. Twenty-four hours thereafter, splenic T cells were isolated, and their IL-2 and IFN- production and MAPK, NF-B, and AP-1 activation were measured. T-cell IL-2 and IFN- production was decreased following trauma-hemorrhage, and this was accompanied with a decrease in T-cell MAPK, NF-B, and AP-1 activation. PPT or 17-estradiol administration following trauma-hemorrhage normalized those parameters, while DPN administration had no effect. Since PPT, but not DPN, administration following trauma-hemorrhage was as effective as 17-estradiol in preventing the T-cell suppression, it appears that ER- plays a predominant role in mediating the salutary effects of 17-estradiol on T cells following trauma-hemorrhage, and that such effects are likely mediated via normalization of MAPK, NF-B, and AP-1 signaling pathways. shock; MAPK; NF-B; activator protein-1; propyl pyrazole triol; diarylpropionitrile     

Inhibition of immune cell proliferation and cytokine production by lipoprotein-bound gangliosides

We have analyzed the immunomodulatory effect of human melanoma gangliosides bound to serum lipoprotein fractions on normal human immune-competent cells in vitro. Total melanoma gangliosides in micelles inhibited proliferation of peripheral blood mononuclear cells stimulated by various mitogens, modulated lymphocyte surface molecules CD2, CD3, CD4, CD5 and CD8 and inhibited the production of interleukin-1(IL-1), tumor necrosis factor (TNF) and IL-6 by stimulated adherent cells. Most of these effects were abrogated in the presence of serum. Purified serum lipoprotein fractions were tested for their ability to allow or inhibit the immunomodulatory effects of gangliosides. Melanoma gangliosides bound to very-low-density lipoproteins (VLDL) were shown to be as potent modulators of the immune response in vitro as when they were presented to cells in the form of micelles. Gangliosides bound to low-density lipoproteins were less active and gangliosides bound to high-density lipoproteins or the lipoprotein-free fraction had no immunomodulatory effects. Given the fact that gangliosides are predominantly bound to lipoproteins in serum, we conclude that lipoproteins are important determinants of the immunomodulating potential of tumor gangliosides, and that the immunomodulatory effects of melanoma gangliosides observed in vitro may also occur in vivo.This study was supported in part by a grant from the FEGEFLUC to J. Portoukalian.     

UVB irradiation suppresses cytokine production and innate cellular immune functions in mice

We examined whether ultraviolet-B (UVB) irradiation (6 kJ/m2) alters cytokine production and other innate immune reactions by murine peritoneal macrophages and peripheral neutrophils. Along with these experiments, serum IgG levels were also assessed. In addition, using scanning electron microscopy (SEM) we observed macrophages that had been exposed to UVB in vitro. Results showed that UVB irradiation: (1) decreased IL-12 production while increasing IL-1alpha secretion from macrophages, but had no effect on IL-1alpha from neutrophils; (2) suppressed phagocytosis of macrophages but not of neutrophils; (3) diminished active oxygen production of macrophages but not of neutrophils; (4) had no effect on serum IgG levels; and (5) caused significant cell destruction of macrophages in vitro. These results suggested: (1) that UVB irradiation could induce characteristic suppression of innate immunity; (2) that innate cellular immunity was more susceptible to the effects of UVB irradiation than humoral immunity.     

Mechanism of the salutary effects of 17beta-estradiol following trauma-hemorrhage: direct downregulation of Kupffer cell proinflammatory cytokine production

Kupffer cells have been reported as a major source of proinflammatory cytokines (i.e. IL-6, TNF-alpha), which have been implicated in the pathogenesis of trauma-hemorrhage. Previous studies have shown a protective effect of 17beta-estradiol on immune function and physiological responses following trauma-hemorrhage. In this study, we investigated whether 17beta-estradiol has a direct effect on Kupffer cell cytokine production following trauma-hemorrhage. Male Sprague-Dawley rats were subjected to trauma (midline laparotomy) and hemorrhage (35-40 mmHg for 90 min followed by fluid resuscitation) or sham operation. Two hours later, Kupffer cells were isolated and cultured with 17beta-estradiol in the presence and absence of lipopolysaccharide stimulation. Kupffer cell IL-6 and TNF-alpha production increased following trauma-hemorrhage. Incubation with 17beta-estradiol attenuated the production of IL-6 by cells from both sham and trauma-hemorrhage animals in a dose-dependent manner. The suppression of IL-6 production by 17beta-estradiol was paralleled by a decrease in mRNA levels. In contrast to IL-6, the effects of 17beta-estradiol on TNF-alpha production were minimal. In conclusion, these results indicate the direct downregulation of Kupffer cell IL-6 production by 17beta-estradiol at a molecular level, which might explain in part the previously observed salutary effects of estradiol treatment following trauma-hemorrhage.     

c-Fos as a regulator of degranulation and cytokine production in FcepsilonRI-activated mast cells

The AP-1 complex is composed of c-Jun and c-Fos and is a key component in the regulation of proinflammatory genes. Mast cells play a significant role in the initiation of many inflammatory responses, such as allergy and allergy-associated diseases. In the present work, we characterized the role of c-Fos in mast cell function by investigating IL-3-dependent cell proliferation, degranulation capability, and cytokine expression in c-Fos-deficient mice. In c-Fos-deficient mast cells, we found that FcepsilonRI-mediated degranulation was significantly inhibited, which correlates with the reduced expression of SWAP-70, VAMP-7, and Synaptotagmin I genes, which are involved directly in the degranulation process. These findings show that c-Fos plays an important role in FcepsilonRI-mediated regulation of mast cell function.     

Ipomoea obscura ameliorates cyclophosphamide-induced toxicity by modulating the immune system and levels of proinflammatory cytokine and GSH

Ipomoea obscura L. is a widely used medicinal plant. The objective of this study was to investigate its protective activity against cyclophosphamide (CTX)-induced toxicity in mouse models. Swiss albino mice were treated intraperitoneally with CTX (25 mg/kg body weight) along with I. obscura extract (10 mg/kg body weight) for 10 days. Extract significantly reduced myelosuppression caused by CTX and improved the relative organ weight, total white blood cell count, and bone marrow cellularity. The elevated levels of parameters related to pathophysiology of the liver, namely glutamate pyruvate transaminase, alkaline phosphatase, and lipid peroxidation, were significantly reduced by extract treatment. Reduction of liver and intestinal glutathione levels of CTX-treated animals was reversed by I. obscura. The lowered levels of cytokines, namely IFN-γ, IL-2, and granulocyte-monocyte colony-stimulating factor after CTX treatment were found to be increased in I. obscura treated animals. Treatment with I. obscura could also decrease the level of proinflammatory cytokine TNF-α. The data suggested that I. obscura can act as a potent chemoprotective agent and can be used as an adjuvant in chemotherapeutic applications.     

Propolis and some of its constituents down-regulate DNA synthesis and inflammatory cytokine production but induce TGF-beta1 production of human immune cells

Propolis, the resinous product collected by honey bees from plants, is used as folk medicine since ancient time. Recently, immunoregulatory and anti-inflammatory properties of propolis have been published. The detailed mechanisms of actions of propolis and its components on immune cells, however, are still unknown. Therefore, we studied the effects of different propolis extracts, of the flavonoids hesperidin and quercetin as well as of caffeic acid phenethyl ester (CAPE) on basic human immune cell functions. In detail, we measured the effects on DNA synthesis and production of different types of cytokines, namely IL-1beta, IL-12, IL-2, IL-4, IL-10 and TGF-beta1, of mitogen-activated peripheral blood mononuclear cells (PBMC) as well as of purified T lymphocytes. Our data clearly show that propolis as well as its constituents studied are capable of dose-dependently suppressing phythemagglutinin (PHA)-induced DNA synthesis of PBMC and T cells. Moreover, cytokines produced by monocytes/macrophages (IL-1beta, IL-12), by Th1 type (IL-2) as well as Th2 type (IL-4) lymphocytes were found to be also suppressed, whereas the production of TGF-beta1 by T regulatory cells was ascertained to be increased. These data convincingly demonstrate that propolis has a direct regulatory effect on basic functional properties of immune cells which may be mediated by the Erk2 MAP-kinase signal pathway. Thus, the bee product propolis can be considered as a powerful natural anti-inflammatory medicine influencing different types of immune-responses probably via immunoregulatory T cells.