Secretions of interleukin-1beta and tumor necrosis factor alpha by whole fetal membranes depend on initial interactions of amnion or choriodecidua with lipopolysaccharides or group B streptococci

The present study evaluated the secretions of interleukin (IL)-1beta and tumor necrosis factor (TNF) alpha by fetal membranes stimulated with group B streptococci (GBS) and lipopolysaccharide (LPS). The aim was to evaluate the initial response of full-thickness membranes to the microbial insult using an in vitro experimental model that allowed testing of the individual contributions of amnion and choriodecidua to stimulation. Full-thickness membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation without evidence of active labor. The membranes were mounted in Transwell devices, physically separating the upper and lower chambers. The LPS (500 ng/ml) or GBS (1 x 10(6) colony-forming units/ml) was added to either the amniotic or choriodecidual surface, and accumulation of IL-1beta and TNFalpha were measured in both compartments using a specific ELISA. Fetal membranes followed different patterns of secretion of proinflammatory cytokines that depended on the side to which the stimulus was added or the nature of the stimulus itself. The TNFalpha was secreted by amnion and choriodecidua in the presence of LPS or GBS, and stimulation with GBS induced a greater synthesis of IL-1beta than did stimulation with LPS. Choriodecidual tissue was more responsive than amniotic tissue, and this response tended to be higher even when the stimulation was only on the amniotic side. However, the amnion plays an active role in recognizing LPS or GBS, contributing a significant amount of TNFalpha. Thus, cooperative and bidirectional communications occur between amnion and choriodecidua in response to bacterial products, which include intermembranous cytokine traffic and signaling between tissues.     

Immunolocalization of proinflammatory cytokines in myometrium, cervix, and fetal membranes during human parturition at term.

Each of the proinflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF) alpha has been identified in reproductive tissues during labor. The cellular origin of these cytokines is unclear. The aim of this study was to localize these proinflammatory cytokines in myometrium (upper and lower segment), cervix, and fetal membranes at term. Biopsies were taken from women undergoing cesarean section either before or after the onset of labor. Immunohistochemistry was used to localize each of the cytokines IL-1beta, IL-6, IL-8, and TNFalpha. Leukocytes were localized using an antibody to CD45. In myometrium and cervix, immunostaining for IL-1beta was predominantly in leukocytes. In fetal membranes, IL-1beta localized to leukocytes and to the stromal cells of the decidua. In myometrium, IL-6, IL-8, and TNFalpha were restricted to leukocytes, which were present in greater numbers in tissue obtained during labor. In cervix, IL-6, IL-8, and TNFalpha localized to leukocytes and glandular and surface epithelium. IL-8 also localized to cervical stromal cells. In fetal membranes, IL-6 and TNFalpha were expressed by decidual stromal cells, infiltrating leukocytes, and extravillous trophoblasts. In membranes, IL-8 localized to leukocytes in the chorion but was not detected in the amnion. In fetal membranes collected at labor, IL-8 was expressed in decidual stromal cells. Infiltrating leukocytes are a major source of cytokines in uterine tissues during labor.     

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.     

Expression of the phospholipid scramblase (PLSCR) gene family during the acute phase response

Phospholipid scramblase 1 (PLSCR1) is a member of PLSCR gene family that has been implicated in multiple cellular processes including movement of phospholipids, gene regulation, immuno-activation, and cell proliferation/apoptosis. In the present study, we identified PLSCR1 as a positive intracellular acute phase protein that is upregulated by LPS in liver, heart, and adipose tissue, but not skeletal muscle. LPS administration resulted in a marked increase in PLSCR1 mRNA and protein levels in the liver. This stimulation occurred rapidly (within 2 h), and was very sensitive to LPS (half-maximal response at 0.1 microg/mouse). Moreover, two other APR-inducers, zymosan and turpentine, also produced significant increases in PLSCR1 mRNA and protein levels, indicating that PLSCR1 was stimulated in a number of models of the APR. To determine signaling pathways by which LPS stimulated PLSCR1, we examined the effect of proinflammatory cytokines in vitro and in vivo. TNFalpha, IL-1beta, and IL-6 all stimulated PLSCR1 in cultured Hep B3 hepatocytes, whereas only TNFalpha stimulated PLSCR1 in cultured 3T3-L1 adipocytes, suggesting cell type-specific effects of cytokines. Furthermore, the LPS-stimulated increase in liver PLSCR1 mRNA was greatly attenuated by 80% in TNFalpha and IL-1beta receptor null mice as compared to wild-type controls. In contrast, PLSCR1 levels in adipose tissue were induced to a similar extent in TNFalpha and IL-1beta receptor null mice and controls. These results indicate that maximal stimulation of PLSCR1 by LPS in liver required TNFalpha and/or IL-1beta, whereas the stimulation of PLSCR1 in adipose tissue is not dependent on TNFalpha and/or IL-1beta. These data provide evidence that PLSCR1 is a positive intracellular acute phase protein with a tissue-specific mechanism for up-regulation.     

Stimulation of interleukin-6 release by interleukin-1beta from isolated human adipocytes

The secretion of interleukin-6 (IL-6) is modulated by immune, hormonal and metabolic stimuli in a cell-specific manner. We investigated the effect of cytokines, TNFalpha and IL-1beta, and insulin on IL-6 release from human adipocytes and peripheral blood cells (PBC). Adipocytes released IL-6 constitutively (after 5 h: 5.64 [1.61-15.30]pg ml(-1), after 10 h: 15.95 [2.34-45.59]pg ml(-1), p = 0.007), while PBC secretion did not change significantly over this period. LPS stimulated IL-6 secretion in PBC after 5 h but was without effect on adipocytes. TNFalpha and insulin induced IL-6 production from PBC, but had no effect on adipocytes. IL-1beta, however, induced a substantial increase in IL-6 release in adipocytes and PBC (all p < 0.05). Adipose tissue production of IL-1beta was assessed in vivo by measuring arterio-venous differences across the subcutaneous abdominal adipose bed. Net release of IL-1beta was not observed, suggesting that under basal conditions there is no detectable release of this cytokine into the circulation from this depot. In conclusion (1) PBC demonstrate regulated IL-6 release, while the adipocyte release has a large constitutive component; (2) immune modulators, such as LPS, TNFalpha and IL-1beta, all induce PBC IL-6 release, but only IL-1beta stimulates adipocyte release. Though IL-1beta is not an endocrine signal from adipose tissue, it is an autocrine/paracrine stimulator of IL-6 release from human adipocytes.     

Local stimulation of prostaglandin production by corticotropin-releasing hormone in human fetal membranes and placenta

Corticotropin-releasing hormone is produced by the human placenta and fetal membranes, but its physiological significance is not established. We examined the possibility that CRH might affect prostaglandin output by these intra-uterine tissues. Primary cultures of amnion, chorion, decidua and placenta were established from tissue obtained from women at term elective cesarean section were maintained in the presence of increasing concentrations of synthetic hCRH. PG output at 48h was measured by radioimmunoassay. hCRH stimulated PGE2 output by amnion, chorion and placenta, but not by decidual tissue. PGF2 alpha output was stimulated in amnion, decidua and placenta but not chorion, whereas output of 13, 14-dihydro-15-keto PGF2 alpha was stimulated in all four tissues. We conclude that hCRH stimulates prostaglandin output by human placenta, decidua and the fetal membranes, raising the possibility of paracrine or autocrine interactions between CRH and prostaglandins in vivo.     

Role of TGF-β1 and TNF-α in IL-1β mediated activation of proMMP-9 in pulmonary artery smooth muscle cells: involvement of an aprotinin sensitive protease

We investigated the role of TGF-β1 and TNF-α in mediating the effect of IL-1β in activating proMMP-9 and proMMP-2, and the involvement of an aprotinin sensitive protease in this scenario in bovine pulmonary artery smooth muscle cells. IL-1β induces TGF-β1 mediated stimulation of 92 kDa proMMP-9 and 72 kDa proMMP-2 mRNA and protein expression; whereas, the elevated level of TNF-α promotes activation of proMMP-9 and proMMP-2. Interestingly, TNF-α induced activation of proMMP-9 appeared to be mediated via a 43 kDa aprotinin sensitive protease. TNF-α inhibited aprotinin and TIMP-1 mRNA and protein expression, which apparently facilitated the proteolytic conversion of proMMP-9 to MMP-9 with the involvement of the aprotinin sensitive protease. The aprotinin sensitive protease did not activate proMMP-2 under IL-1β stimulation, albeit a marked inhibition of TIMP-2 mRNA and protein expression were elicited by TNF-α. Thus, IL-1β induced stimulation of the two progelatinases occurs via different mechanisms.     

The expression of interleukin-15 and interleukin-18 by human term placenta is not affected by lipopolysaccharide

The aim of the study was to examine the stimulatory effect of the inflammatory agent lipopolysaccharide (LPS) on the capacity of human term placenta to secrete interleukin (IL)-15 and IL-18. Isolated placental cotyledons from normal human term deliveries were dually perfused for ten hours with perfusion medium alone (n=5) or with perfusion medium containing LPS (1 microg/kg perfused placental tissue) (n=5). Placental tissue was collected from three different placental compartments (amnion, chorion, and placenta) before and after perfusion. The placental tissues collected were homogenized and examined for IL-15 and IL-18 by ELISA. In addition, formalin-fixed and paraffin-embedded sections from term placentas before perfusion were stained by immunohistochemistry to characterize the cellular origin of placental IL-15 and IL-18. Statistical significance was determined using paired/unpaired t-test. p<0.05 was considered significant. Our results show that IL-15 and IL-18 are produced more by chorionic tissue, as compared to the amnion and placental tissues. Moreover, we show that IL-15 and IL-18 are expressed by epithelial cells of the amnion, chorionic cells of the chorion and decidual cells of the decidua. However, IL-15, but not IL-18, was expressed also by syncytiotrophoblasts of the villi. Perfusion of LPS did not affect the capacity of amnion, chorion and placental tissues to secrete IL-15 and IL-18, as compared to control. The expression of IL-15 and IL-18 in the different compartments of the human placenta suggests a possible role for these two cytokines in normal placental development, pregnancy and labor. Moreover, our results indicate that IL-15 and IL-18 are not part of the mechanism of the response of human placenta to LPS.     

Lipopolysaccharide induces the expression of interleukin-1alpha distinctly in different compartments of term and preterm human placentae

The aim of the study was to investigate the stimulatory effect of lipopolysaccharide (LPS) on IL-lalpha production in different compartments of term and preterm placental tissues. Homogenates from amnion, chorion, and from fetal (subchorionic placental tissues, maternal decidua, and mid-placental tissue before and after perfusion of isolated placental cotyledons of 5 term placentas and 4 placentas obtained after preterm birth (28-34 W of gestation) were examined. Isolated placental cotyledons were dually perfused LPS (100 ng/kg perfused placental tissue) was perfused into the maternal side during 10 hours. Homogenates of the samples were examined by ELISA for IL-1alpha levels, and paraffin sections of the samples were stained by immunohistochemical staining, to characterize the cellular origin of placental IL-1alpha. Paired t test and ANOVA determined statistical significance. In the homogenates, there was a tendency towards higher IL-lalpha levels in all preterm placental compartments as compared to the term compartments before perfusion. A significant increase was observed only in the chorion compartment (p = 0.035). LPS had significantly increased IL-la levels only in the decidua compartment of term placentas as compared to other placental compartments (p = 0.0004), and had decreased IL-1alpha levels in the mid-placenta (p = 0.034). In preterm placentas, addition of LPS did not affect the expression levels of IL-1alpha in either fetal or maternal compartments as determined by ELISA and immunohistochemical staining. IL-la levels in the chorion compartment of preterm placenta were significantly higher as compared to term placenta. LPS affects placental tissues of term and preterm placentas differently. Also, in the term placentas, LPS affected the different compartments differently. Thus, IL-1alpha may have a key role (as a autocrine/paracrine factor) in the regulation of normal and pathological pregnancy and parturition.     

Dynamics of cytokine production in human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia

To specify the role of individual cytokines in the immune response to pyrogens, isolated and cultivated human peripheral blood mononuclear cells (PBMC) were used for the experiments. Different pyrogens (lipopolysaccharide from Escherichia coli - LPS and live Borrelia afzelii) were applied and the time course of changes in concentrations of different cytokines in the medium was followed using the ELISA method. It was found that nonstimulated human PBMC proliferate under in vitro conditions and produce IL-6, TNF-alpha, IL-10 and finally also IL-1beta. Productions of IL-12 and INF-gamma are not changed. Proliferation of PBMC is potentiated after incubation with LPS or live Borrelia. PBMC stimulated by LPS increase the net production (stimulated minus unstimulated) of IL-1beta and TNF-alpha significantly, while production of IL-6 was smaller. A delayed increase in the production of IL-10 was also observed. Productions of IL-12 and INF-gamma were not influenced. In contrast to LPS, stimulation of PBMC with live Borrelia, increases also the production of IL-12 and IFN-gamma, besides IL-1beta, TNF-alpha, IL-6 and IL-10. Productions of IL-1beta, IL-6 and TNFalpha increased immediately after incubation with both LPS and Borrelia, while productions of IL-12 and INF-gamma begin to increase 8 hours and production of IL-10 12 hours after stimulation. Data indicate that stimulation with different pyrogens may activate the cells of the immune cascade in a different way. Stimulation of BPMC by LPS seems to activate the initial steps of the immune response (macrophages and granulocytes) only, while infection with live Borrelia also stimulates the later phase of the immune response, probably due to effect of initially produced cytokines.     

Autocrine growth factors secreted by the malignant human B-cell-line BJAB are distinct from other known cytokines

BJAB, a EBV-negative Burkitt-like lymphoma, did not grow under suboptimal culture conditions in low concentrations of serum unless appropriate cytokines were added. A subclone of BJAB, Clone 13, however, could be kept in long-term culture under such conditions without added cytokines. This suggested that growth of BJAB-Clone 13 was supported by autocrine growth factors (AGF). In fact, the supernatant of Clone 13 stimulated growth of the parental BJAB line and showed IL-1-like activity. Of several cytokines tested only AGF and IL-1 stimulated growth of BJAB. IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, GM-CSF, TNF-alpha, LT, IFN-gamma and TGF beta did not have this effect. The IL-1-like activity was completely neutralized by anti-IL-1 alpha antibodies. In contrast, AGF-activity was not affected by anti-IL-1 alpha. Rabbit antibodies produced against fractions enriched for AGF inhibited growth of BJAB. This inhibition was overcome by Clone 13-AGF, but not by IL-1 alpha. These data suggest that Clone 13-AGF is distinct from IL-1 alpha and might be a new cytokine.     

Regulation of pro-inflammatory cytokine expression by curcumin in hyaline membrane disease (HMD)

Persistent expression of pro-inflammatory cytokines is believed to play a major role in the pathogenesis of chronic lung disease (CLD) in premature infants. Inhibition of pro-inflammatory cytokine production in the lungs of preterm newborns may result in the attenuation of CLD. Curcumin is a naturally occurring phenolic compound derived from the food spice tumeric with broad based in vitro anti-inflammatory properties. In this study lung inflammatory cells from preterm newborns at risk for the development of CLD were derived via modified broncho-alveolar lavage and stimulated ex vivo with lipopolysaccharide (LPS) (10 ng/ml). Curcumin was added to these cultures at 0, 0.5 and 20 uM concentrations. Pro-inflammatory cytokine, TNFalpha, IL-1beta and IL-8 protein was measured from the culture supernatants 12 hours post culture. For control, adult peripheral blood mononuclear cells (PBMC) were cultured under the same conditions. Both neonatal lung inflammatory cells and adult PBMC produced high levels of pro-inflammatory cytokines in response to LPS. Curcumin produced significant inhibition of IL-1beta and IL-8 but minimal inhibition of TNFalpha expression by preterm lung inflammatory cells at 20 uM concentrations. Adult PBMC expression of IL-8 was significantly inhibited by curcumin at 20 uM concentrations. Therefore, curcumin inhibits pro-inflammatory cytokine production (TNFalpha, IL-1beta and IL-8) by lung inflammatory cells ex vivo. Pathways involved with curcumin regulation of these cytokines are developmentally intact and functional in premature infants. Curcumin may be effective as a therapeutic agent in the attenuation of CLD.     

Interleukin 4 and 13 participation in mycobacterial (type-1) and schistosomal (type-2) antigen-elicited pulmonary granuloma formation: multiparameter analysis of cellular recruitment, chemokine expression and cytokine networks

The contribution of IL-4 and IL-13 to inflammation and cytokine responses was compared in mice with types-1 or -2 pulmonary granulomas (GR) elicited by beads bound to antigens of Mycobacteria bovis (PPD) or Schistosoma mansoni eggs (SEA). Type-2 SEA-GR produced the most IL-4 and IL-13. Type-1 PPD-GR produced detectable IL-13, but not IL-4. Mice were treated with anti-IL4 or anti-IL-13 Abs, then lesion size/composition, cytokine/chemokine mRNA and lymph node cytokines were measured. Type-1 GRs resisted individual Abs, but combined Abs augmented lesions by 20%. In contrast, anti-IL-4 abrogated type-2 GR by 30-40% and eosinophil recruitment by 60%. Anti-IL-13 abrogated type-2 GR by 20-30% with no effect on eosinophils. Combined depletion reduced lesion area by 60% and eosinophils by more than 80%. In type-1 GR lungs, anti-IL-4 and anti-IL-13 augmented IFNgamma and TNFalpha mRNA. In type 2 lungs, anti-IL-13 did likewise, but anti-IL-4 decreased TNFalpha without affecting IFNgamma mRNA. In both responses, IL-4 promoted MCP-1 and MCP-5 mRNA, but IL-13 inhibited chemokines in type-1 GR. In lymph nodes, anti-IL-4, but not anti-IL-13, abrogated type-2 cytokines. In fact, IL-13 down-regulated itself and other type-2 cytokines. In summary, IL-4 and IL-13 have common and disparate regulatory functions in types 1 and 2 responses.     

Regulation of human monocyte proMMP-9 production by fetuin, an endogenous TGF-beta antagonist

Members of the matrix metalloproteinase family of enzymes degrade specific components of the extracellular matrix. MMP-9 is a type IV/V collagenase necessary for normal osteogenesis and is increased in inflammatory and neoplastic conditions. In such destructive diseases as emphysema and arthritis, and in epithelial cancers, MMP-9 is produced by cells of the monocyte lineage. Fetuin, a prominent serum glycoprotein, binds to and inactivates TGF-beta family members and through this mechanism regulates osteogenesis (Binkert et al., 1999, J Biol Chem 274:28514-28520.). We studied the effects of TGF-beta1 and fetuin on proMMP-9 release by the human monocyte line THP-1. Exogenous TGF-beta1 stimulated proMMP-9 release by THP-1 cells, with half-maximal stimulation at approximately 0.01 ng/ml TGF-beta1. Human fetuin (0.5-2 microM) partially inhibited this stimulation. Human fetuin alone stimulated THP-1 monocyte proMMP-9 release, with half maximal stimulation at approximately 0.25 microM fetuin. Neutralizing antibody specific for TGF-beta1 also stimulated proMMP-9 release, suggesting that endogenously-derived TGF-beta1 has an inhibitory effect. In freshly isolated human peripheral blood monocytes, fetuin stimulated proMMP-9 release with a dose-response curve similar to that observed in THP-1 cells. Human fetuin also activated proMMP-9 present in THP-1 conditioned medium. Taken together, these data suggest that under physiological conditions, fetuin facilitates matrix degradation by monocyte-derived MMP-9, both by opposing the autocrine inhibitory effect of endogenous TGF-beta1 on proMMP-9 release, and by activating proMMP-9 in the pericellular milieu. Conversely, fetuin may limit the stimulation of monocyte proMMP-9 release by high levels of exogenous TGF-beta1. Such modulation could prove important under pathological conditions where TGF-beta1 derived from paracrine sources is abundant, such as in epithelial malignancies.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

IL-6 and tumor necrosis factor-alpha. Autocrine and paracrine cytokines involved in B cell function

IL-6 and TNF-alpha are synthesized and secreted by normal tonsillar B cells after stimulation with the polyclonal B cell activator Staphylococcus aureus Cowan strain 1 (SAC) and IL-2 as well as spontaneously by in vivo activated B cells from patients with hypergammaglobulinemia. Using specific neutralizing antibodies, both factors were shown to be involved in autocrine and/or paracrine regulation of B cell differentiation. IgG induced by SAC/IL-2 stimulation was reduced 73% with an anti-IL-6 antibody and 40% with an anti-TNF-alpha antibody. Similar effects of these antibodies were observed on the spontaneous in vitro IgG production by lymphoblastic B cells from six patients with hypergammaglobulinemia. Kinetic studies with SAC/IL-2-activated B cells revealed that the anti-TNF-alpha antibody must be present at the beginning of the culture to exert an effect on Ig production, whereas the anti-IL-6 antibody reduced Ig production even if added as late as day 3. This sequential action of TNF-alpha and IL-6 on B cell differentiation was reflected by different kinetics of release of these two cytokines into the supernatant of SAC/IL-2 activated B cells; TNF-alpha peaked at 24 h and IL-6 at 96 h after stimulation. In addition, it was shown that IL-6 production by in vitro-activated B cells was partially blocked by an anti-TNF-alpha antibody suggesting that TNF-alpha regulates IL-6 production in normal B cells via an autocrine pathway. We also investigated the effects of TGF-beta on TNF-alpha and IL-6 production by normal B cells. Although TGF-beta inhibited Ig production by in vitro-activated and in vivo-activated B cells, it did not inhibit the release of these cytokines from normal B cells. Furthermore, TGF-beta did not inhibit the induction of nuclear factor-IL-6 nor the expression of IL-6R on activated B cells. Thus, although the biologic effects of anti-IL-6 and TGF-beta on B cell Ig production are similar, their mechanisms of actions appear to be distinct.     

Estradiol receptor potentiates,in vitro,the activity of 5-methylcytosine DNA glycosylase

Heme oxygenase-1 (HO-1) is induced under various oxidative stress conditions, such as lipopolysaccharide (LPS) insult. Induction of HO-1 by LPS is reported to be mediated through interleukin-1beta (IL-1beta), rather than other inflammatory cytokines in the mouse liver. However, we found that IL-1alpha/beta knockout (KO) mice responded well to LPS insult, as did wild-type mice with respect to HO-1 mRNA induction (about 30-fold increase). In contrast, tumor necrosis factor alpha KO (TNFalphaKO) mice responded very weakly to LPS in the HO-1 mRNA expression, but not metallothionein mRNA. Recent studies reveal that nitric oxide from Kupffer cells is involved in HO-1 induction in the liver produced by LPS. Therefore, nitrite and nitrate concentrations in the liver were also measured and these parameters did not increase in either IL-1KO or TNFalphaKO. In addition, the phosphorylation of c-JUN N-terminal kinase (JNK) and p38, but not extracellular signal-regulated kinase, was very low in TNFalphaKO mice due to LPS administration. All of these findings indicate that TNFalpha is a major candidate to trigger HO-1 induction in response to LPS stimulation, and that its message is likely transduced through JNK and p38 pathways.     

Synergistic induction of interleukin-6 production and gene expression in human thymic epithelial cells by LPS and cytokines

We examined the ability of LPS and several cytokines (TNF-alpha, IL-1-beta, IFN-gamma, IL-4) to modulate IL-6 production by cultured human thymic epithelial cells (TEC). IL-6 activity was measured by the hybridoma growth factor biological activity. Moderate but detectable IL-6 activity was spontaneously produced in the presence of serum proteins. LPS as well as the cytokines TNF-alpha and IL-1-beta was a potent inducer of IL-6, increasing, respectively, IL-6 levels by 9-, 28-, and 75-fold (mean values) while IL-4 and IFN-gamma provoked no significant effect. Interestingly, clearly different kinetics were observed for IL-6 induction by the various activation agents, the maximal effect being reached at 24, 48, and 72 hr, respectively for LPS, TNF-alpha, and IL-1-beta. Moreover, a synergistic effect of TNF-alpha and either LPS or IL-1-beta was observed. Indeed, TEC incubated with the cytokines in combination at optimal doses produced 5- to 170-fold more IL-6 than TEC stimulated with the cytokines individually. Neutralizing anti-IL-6 polyclonal and monoclonal antibodies completely blocked hybridoma proliferation stimulating activity of TEC supernatants; thus, implying that this activity is essentially due to IL-6. In situ hybridization analysis of cytocentrifuged TEC with an mRNA antisense probe specific for human IL-6 and labeled with 35S demonstrated that up to 90% of TEC could be induced to express the IL-6 gene. Computer-aided quantification of IL-6 mRNA levels indicated that upon stimulation with TNF-alpha combined to LPS, both the numbers of cells expressing IL-6 mRNA and the amounts of cytoplasmic IL-6 mRNA per cell were increased. Taken altogether these results demonstrate that LPS and/or cytokines can modulate and synergistically stimulate IL-6 production. In addition to a possible role in regulating normal thymic T cell activation, the IL-6 produced by TEC could be of pathophysiological relevance in disregulated situations such as in hyperplastic thymuses from patients with myasthenia gravis.