Cell type-dependent response to benzo(a)pyrene exposure of human placental cell lines under normoxic, hypoxic, and pro-inflammatory conditions.

Benzo(a)pyrene (BaP) can be detected in the human placenta. However, little is known about the effects of BaP exposure on different placental cells under various conditions. In this study, we aimed to investigate the effects of BaP on mitochondrial function, pyrin domain-containing protein 3 (NLRP3) inflammasome, and apoptosis in three human trophoblast cell lines under normoxia, hypoxia, and inflammatory conditions. JEG-3, BeWo, and HTR-8/SVneo cell lines were exposed to BaP under normoxia, hypoxia, or inflammatory conditions for 24 h. After treatment, we evaluated cell viability, apoptosis, aryl hydrocarbon receptor (AhR) protein and cytochrome P450 (CYP) gene expression, mitochondrial function, including mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨm), intracellular adenosine triphosphate (iATP), and extracellular ATP (eATP), nitric oxide (NO), NLPR3 inflammasome proteins, and interleukin (IL)-1ß. We found that BaP upregulated the expression of AhR or CYP genes to varying degrees in all three cell lines. Exposure to BaP alone increased ΔΨm in all cell lines but decreased NO in BeWo and HTR-8/SVneo, iATP in HTR-8/SVneo, and cell viability in JEG-3, without affecting apoptosis. Under hypoxic conditions, BaP did not increase the expression of AhR and CYP genes in JEG-3 cells but increased CYP gene expression in two others. Pro-inflammatory conditions did not affect the response of the 3 cell lines to BaP with respect to the expression of CYP genes and changes in the mitochondrial function and NLRP3 inflammasome proteins. In addition, in HTR-8/SVneo cells, BaP increased IL-1ß secretion in the presence of hypoxia and poly(I:C). In conclusion, our results showed that BaP affected mitochondrial function in trophoblast cell lines by increasing ΔΨm. This increased ΔΨm may have rescued the trophoblast cells from activation of the NLRP3 inflammasome and apoptosis after BaP treatment. We also observed that different human trophoblast cell lines had cell type-dependent responses to BaP exposure under normoxia, hypoxia, or pro-inflammatory conditions.

Early pregnancy biomarker discovery study for spontaneous preterm birth.

(1) OBJECTIVE: discover new candidate biomarkers for spontaneous preterm birth in early pregnancy samples. When fully clinically validated, early pregnancy biomarkers for sPTB give the possibility to intervene or monitor high-risk pregnancies more intensively through, as example, pelvic exams, ultrasound or sonographic cervical length surveillance. (2) STUDY DESIGN: Early pregnancy serum samples of eight spontaneous extreme and very preterm birth cases (<32 weeks of gestational age) without any symptoms of preeclampsia and fetal growth restriction and eight uncomplicated pregnancies were analyzed by liquid chromatography mass spectrometry (LC-MS). Thirteen proteins, which were differentially expressed according to the LC-MS data, were subsequently selected for confirmation by enzyme-linked immunosorbent assay (ELISA). (3) RESULTS: Differential expression of four candidate biomarkers was confirmed by ELISA with decreased early pregnancy levels of gelsolin and fibulin-1 and increased levels of c-reactive protein and complement C5 in the preterm birth group. (4) CONCLUSIONS: The confirmed candidate biomarkers are all to some extent related to inflammatory pathways and/or the complement system. This supports the hypothesis that both play a role in extreme and very preterm birth without any symptoms of preeclampsia and fetal growth restriction. The predictive value of complement C5, c-reactive protein, fibulin-1 and gelsolin should, therefore, be validated in another cohort with early pregnancy samples.

First trimester serum biomarker discovery study for early onset, preterm onset and preeclampsia at term.

INTRODUCTION: Preeclampsia (PE) is a heterogeneous syndrome during pregnancy and postpartum and it is subdivided in this study into early onset (<34 weeks), preterm onset (34-37 weeks) and PE at term (>37 weeks). First trimester models currently lack a sufficient power to predict PE, but inclusion of biochemical markers shows an improvement of their predictive power. The aim of this study was to perform a biomarker discovery study in order to find possible novel first trimester biomarkers for each PE subtype. Further, our findings were related to available literature and the possible role of the proteins in the development of preeclampsia was discussed. METHODS: In this study, 9 early onset (<34 weeks), 8 preterm onset (34-37 weeks), 6 PE at term (>37 weeks) and 23 control samples were drawn between 11 and 14 weeks gestational age. Serum samples were prepared for liquid chromatography mass spectrometry analysis and protein data were exported for statistical analyses. All differentially expressed proteins were further evaluated by searching literature in MEDLINE, Embase and Web of science and differential expression of two proteins, which were not yet associated with PE, was verified through enzyme-linked immunosorbent assay (ELISA). RESULTS: After statistical analysis, six, four and eight proteins were differently expressed in early onset, preterm onset and PE at term, respectively. After exclusion of antibody fragments, only nine proteins remained. Seven out of these nine proteins were already in literature associated with preeclampsia and only three of them were described as differentially expressed in the first trimester or early second trimester of preeclamptic pregnancies. Differential expression of Apolipoprotein D (ApoD), which was not yet associated with PE, was confirmed by ELISA in both early and preterm onset PE in the first trimester. DISCUSSION: In this study, two main observations were made. First, some of the differentially expressed proteins have a role in the same biological pathway, such as the acute phase response or endometrium receptivity, and their differential expression was observed in all three PE subtypes. This observation supports the hypothesis that classification of PE could be more accurate when subtyping is based on the etiology and/or phenotype instead of the arbitrary parameter gestational age at onset or delivery. Second, seven differential expressed proteins were already associated in literature with preeclampsia, but this association was for only three of them observed in the first trimester. In addition, ApoD was not yet associated with PE in other studies and, moreover, its differential expression was confirmed by ELISA. Therefore the predictive power of these proteins in the first trimester is worth evaluating in a larger and more heterogeneous cohort.

Healthy and preeclamptic pregnancies show differences in Guanylate-Binding Protein-1 plasma levels.

The large interferon-inducible anti-angiogenic pro-inflammatory GTPase Guanylate Binding Protein-1 (GBP-1) is produced and secreted by activated endothelial cells and is highly induced by inflammatory cytokines and inhibited by angiogenic growth factors. During pregnancy a generalized mild inflammatory response is observed. During preeclampsia this generalized inflammatory response is even further activated and activation of the endothelium occurs. We hypothesized that GBP-1 is increased in healthy pregnancy and will be even further increased during preeclampsia. In the first experiment, plasma and placentas were collected from healthy and preeclamptic pregnancies. Plasma was also collected from non-pregnant women. For the second experiment longitudinal blood samples from women with a healthy or preeclamptic pregnancy were collected from the end of the first trimester until birth and one sample postpartum. The plasma GBP-1 levels were measured by ELISA and GBP-1 mRNA and protein levels in the placenta were tested by qPCR and immunohistochemistry. During pregnancy higher plasma concentrations of GBP-1 compared with non-pregnant women were observed. Surprisingly, during preeclampsia, plasma GBP-1 levels were lower than in control pregnancies and similar to the level of non-pregnant controls. Placental GBP-1 mRNA levels were not different between healthy and preeclamptic pregnancies and GBP-1 protein was virtually undetectable in the trophoblast by immunohistochemistry in placental tissue. Evaluation of longitudinal samples showed that plasma GBP-1 concentrations increased towards the end of pregnancy in healthy pregnancies, but not in preeclampsia. In line with our hypothesis, we found higher GBP-1 plasma levels during healthy pregnancy. However, plasma GBP-1 did not further increase during preeclampsia, but was stable. Further studies are needed to evaluate why GBP-1 does not increase during preeclampsia.

First trimester secreted Frizzled-Related Protein 4 and other adipokine serum concentrations in women developing gestational diabetes mellitus.

BACKGROUND: The aim of this study was to evaluate whether soluble frizzled-related protein 4 (sFRP4) concentration in the first trimester of pregnancy is individually, or in combination with Leptin, Chemerin and/or Adiponectin, associated with the development of gestational diabetes (GDM). METHODS: In a nested case-control study, 50 women with GDM who spontaneously conceived and delivered a live-born infant were matched with a total of 100 uncomplicated singleton control pregnancies based on body mass index (± 2 kg/m2), gestational age at sampling (exact day) and maternal age (± 2 years). In serum samples, obtained between 70-90 days gestational age, sFRP4, Chemerin, Leptin and Adiponectin concentrations were determined by ELISA. Statistical comparisons were performed using univariate and multi-variate logistic regression analysis after logarithmic transformation of the concentrations. Discrimination of the models was assessed by the area under the curve (AUC). RESULTS: First trimester sFRP4 concentrations were significantly increased in GDM cases (2.04 vs 1.93 ng/ml; p<0.05), just as Chemerin (3.19 vs 3.15 ng/ml; p<0.05) and Leptin (1.44 vs 1.32 ng/ml; p<0.01). Adiponectin concentrations were significantly decreased (2.83 vs 2.94 ng/ml; p<0.01) in GDM cases. Further analysis only showed a weak, though significant, correlation of sFRP4 with Chemerin (R2 = 0.124; p<0.001) and Leptin (R2 = 0.145; p<0.001), and Chemerin with Leptin (R2 = 0.282; p<0.001) in the control group. In a multivariate logistic regression model of these four markers, only Adiponectin showed to be significantly associated with GDM (odds ratio 0.12, 95%CI 0.02-0.68). The AUC of this model was 0.699 (95%CI 0.605-0.793). CONCLUSION: In the first trimester of pregnancy, a multi-marker model with sFRP4, Leptin, Chemerin and Adiponectin is associated with the development of GDM. Therefore, this panel seems to be an interesting candidate to further evaluate for prediction of GDM in a prospective study.

Changes in endothelial cell specific molecule 1 plasma levels during preeclamptic pregnancies compared to healthy pregnancies.

OBJECTIVE: We aimed to assess the levels of endothelial cell specific molecule 1 (ESM-1) during pregnancy and preeclampsia. METHODS: Plasma and placental samples were collected from women with a control pregnancy, early- or late-onset preeclamptic women and non-pregnant women (experiment 1). Plasma samples were collected between weeks 12 and birth from pregnant women at high risk for developing preeclampsia (experiment 2). ESM-1 plasma levels were measured by ELISA and in the placenta mRNA and protein were detected by immunohistochemistry and qPCR. RESULTS: In the first experiment we observed lower concentrations of ESM-1 in pregnant women as compared to non-pregnant women and higher concentrations during early- and late-onset preeclampsia as compared to control pregnancies of the same gestational age. Early- and late-onset preeclamptic pregnancies were not different from their subsequent controls in ESM-1 mRNA or protein levels in placental tissue. The second experiment showed that in women who had an control pregnancy, plasma ESM-1 levels were decreased as compared to non-pregnant women, from week 16 ±â€¯2 until the end of pregnancy and returned to non-pregnant levels postpartum. In women who developed early- or late-onset preeclampsia, plasma ESM-1 was also decreased as compared to non-pregnant women from week 20 ±â€¯2 until week 28 ±â€¯2 of pregnancy. Then ESM-1 levels increased and were no longer different from levels in non-pregnant women on weeks 32 and 36. CONCLUSIONS: Plasma ESM-1 levels are decreased during pregnancy and increased in early- and late-onset preeclampsia. The source of ESM-1 is probably not the placenta, but most likely maternal endothelial cells.

Reference values of fetal erythrocytes in maternal blood during pregnancy established using flow cytometry.

The aim of our study was to assess the fetal RBC count in maternal blood during uncomplicated pregnancies from 26 weeks onward. We used a flow cytometric method specifically designed for use in a routine hematology analyzer. Pregnant women were recruited through midwives. The participating laboratories used the FMH QuikQuant method (Trillium Diagnostics, Brewer, ME) in a CELL-DYN Sapphire hematology analyzer (Abbott Diagnostics, Santa Clara, CA). The method is based on a monoclonal antibody to hemoglobin F. Flow cytometric data were analyzed by 2 independent observers. The 95th percentile reference range was estimated according to Clinical and Laboratory Standards Institute guidelines. A total of 236 samples were statistically analyzed. Gestational ages ranged from 21.6 to 41 weeks (mean, 32.0 weeks), and the fetal RBC count in maternal blood ranged from 0.00% to 0.50% (median, 0.025%). The fetal RBC count in maternal blood shows no correlation with gestational age. The established reference range during normal pregnancy is less than 0.125%.

Novel serum biomarkers in carotid artery stenosis: useful to identify the vulnerable plaque?

OBJECTIVES: Serum biomarkers representing inflammatory activity in vulnerable carotid plaques may be used to identify high-risk patients for cerebral ischemic events. We aimed to analyze the relationship between concentrations of four novel biomarkers and neurological symptoms: Neopterin, PTX3, sCD163, and sTREM-1. In addition, we analyzed the relationship between these markers and the presence of coronary (CAD) and peripheral (PAD) artery disease. DESIGN AND METHODS: Serum biomarker levels were determined in 100 patients undergoing carotid endarterectomy; 33 for stroke, 32 for transient ischemic attack, and 23 for amaurosis fugax. 12 Patients were asymptomatic. Risk factors for atherosclerotic disease and history of CAD and PAD were also assessed. RESULTS: Symptomatic patients did not show significantly elevated biomarker levels compared to asymptomatic patients and levels did not differ among symptomatic subgroups. Neopterin levels were elevated in patients with concomitant coronary and peripheral artery disease (CAD (32%) 10.2 ± 6.6 vs no CAD (68%) 7.6 ± 2.9 nmol/L, PAD (20%) 12.3 ± 7.4 vs no PAD (80%) 7.5 ± 3.0 nmol/L, p<0.05). sTREM-1 was elevated in patients with CAD (50.8 ± 53.2 vs 28.0 ± 31.6 ng/L, p<0.05). PTX3 and sCD163 were not significantly elevated in CAD nor PAD. CONCLUSION: Our findings suggest that serum neopterin and sTREM-1 levels may be related to the presence of atherosclerotic disease, but not to carotid plaque vulnerability.

Noncanonical NF-kappaB signaling in dendritic cells is required for indoleamine 2,3-dioxygenase (IDO) induction and immune regulation.

Ligation of CD40 on dendritic cells (DCs) induces early production of inflammatory mediators via canonical NF-kappaB signaling, as well as late expression of the anti-inflammatory enzyme indoleamine 2,3-dioxygenase (IDO) via unknown signal transduction. By selective blocking of either the canonical NF-kappaB pathway using the NEMO-binding domain peptide or the noncanonical NF-kappaB pathway by small interfering RNA, we demonstrate that IDO expression requires noncanonical NF-kappaB signaling. Also, noncanonical NF-kappaB signaling down-regulates proinflammatory cytokine production in DCs. In addition, selective activation of the noncanonical NF-kappaB pathway results in noninflammatory DCs that suppress T-cell activation and promote the development of T cells with regulatory properties. These findings reveal an important role of the noncanonical NF-kappaB pathway in the regulation of immunity.

Human keratinocytes express functional Toll-like receptor 3, 4, 5, and 9.

Keratinocytes are continuously in contact with external stimuli and have the capacity to produce several soluble mediators. Pathogen-associated molecular patterns (PAMPs) are recognized, among others, by Toll-like receptors (TLRs). The functional responses of keratinocytes to different PAMPs have not yet been fully established. Here we show that keratinocytes constitutively express TLR1, 2, 3, 4, 5, 6, 9, and 10 mRNA, but not TLR7 and 8. Stimulation of keratinocytes with TLR3, 4, 5, and 9 ligands resulted in differential immune-associated responses. Tumor necrosis factor-alpha, CXC chemokine ligand 8 (CXCL8), CCL2, and C chemokine ligand 20 (CCL20) release was enhanced in response to all PAMPs tested, in a time- and dose-dependent manner. Only TLR9 ligand CpG-oligodeoxynucleotides (ODNs) and TLR3 ligand poly-I:C could additionally induce type I IFNs. CCL27 production was selectively induced by poly-I:C and flagellin, whereas CXCL9 and CXCL10 were exclusively induced by CpG-ODNs and/or poly-I:C. Upregulation of ICAM-1, HLA-DR, HLA-ABC, FasR, and CD40 was mainly observed in response to poly-I:C, flagellin, and lipopolysaccharide. Furthermore, PAMP triggering resulted in the phosphorylation of phosphorylated-IkappaB alpha and in the nucleus translocation of NF-kappaB p65. Altogether, these findings stress an unexpectedly multifaceted role of keratinocytes in innate immunity as evident by their differential, TLR-mediated responses to PAMPs associated with different classes of pathogens.

Analysis of anthrax and plague biowarfare vaccine interactions with human monocyte-derived dendritic cells.

The anti-biowarfare anthrax and plague vaccines require repeated dosing to achieve adequate protection. To test the hypothesis that this limited immunogenicity results from the nature of vaccine interactions with the host innate immune system, we investigated molecular and cellular interactions between vaccines, dendritic cells (DCs), and T cells and explored the potential for adjuvants (pertussis) to boost induction of host immunity. Human monocyte-derived DCs were matured in the presence of vaccines and analyzed for their ability to induce Th1/Th2 development from naive T cells, expression of cell surface maturation/costimulation molecules, and cytokine production. The vaccines showed different behavior patterns. Although the plague vaccine is equivalent to control maturation factors in maturation and stimulation of DCs and induces strong MLR and Th outgrowth, the anthrax vaccine is a poor inducer of DC maturation, as indicated by low levels of HLA-DR, CD86, and CD83 induction and minimal proinflammatory cytokine production. Interestingly, however, anthrax vaccine-treated DCs stimulate Th1 and Th2 outgrowth and a limited MLR response. There was no sustained negative modulatory effects of the anthrax vaccine on DCs, and its limited stimulatory effects could be overridden by coculture with pertussis. These results were supported by analysis of anthrax vaccine recall responses in subjects vaccinated using pertussis as an adjuvant, who demonstrate anthrax-specific effector T cell responses. These data show that the anthrax vaccine is a suboptimal DC stimulus that may in part explain the observation that it requires repeated administration in vivo and offer a rational basis for the use of complementary DC-maturing adjuvants in combined immunotherapy.

Double-stranded RNA-exposed human keratinocytes promote Th1 responses by inducing a Type-1 polarized phenotype in dendritic cells: role of keratinocyte-derived tumor necrosis factor alpha, type I interferons, and interleukin-18.

Dendritic cells play a key role in establishing the class of immune response against invading pathogens. Upon engagement with double-stranded RNA, a major bioactive constituent of many virus types, immature dendritic cells develop into type 1 immunostimulatory dendritic cells that promote Th1 responses. Immature dendritic cells reside in the epithelia and are in close contact with keratinocytes. We studied to what extent dendritic cells can also adopt a type 1 immunostimulatory dendritic cell phenotype indirectly, as a result of the interaction with keratinocytes responding to double-stranded RNA. In contrast to supernatants from keratinocytes activated by the combination of tumor necrosis factor alpha and interleukin-1beta, supernatants from keratinocytes activated by synthetic double-stranded RNA, polyriboinosinic polyribocytidylic acid, comprised tumor necrosis factor alpha and type I interferons, which induced maturation of human monocyte-derived immature dendritic cells. In addition, dendritic cells matured in the presence of these supernatants strongly biased the development of Th1 cells from naive Th cells. This bias was dependent on keratinocyte-derived interferon-alpha/beta and interleukin-18, as neutralization of both interferon-alpha/beta and interleukin-18 in the keratinocyte culture supernatant reduced the development of interferon-gamma-producing Th cells. These findings suggest that keratinocytes can contribute to the development of selective Th1/Th2 responses through the induction of maturation and functional polarization of dendritic cells, indicating a novel role for keratinocytes as initiators and regulators of cutaneous T-cell-mediated inflammation. In addition, these results support the concept that, in addition to direct interaction with pathogens, dendritic cells may also be activated and primed by pathogen indirectly, via the effect of resident tissue cells responding to pathogen.

Differential transmission of human immunodeficiency virus type 1 by distinct subsets of effector dendritic cells.

Dendritic cells (DC) support human immunodeficiency virus type 1 (HIV-1) transmission by capture of the virus particle in the mucosa and subsequent transport to the draining lymph node, where HIV-1 is presented to CD4(+) Th cells. Virus transmission involves a high-affinity interaction between the DC-specific surface molecule DC-SIGN and the viral envelope glycoprotein gp120 and subsequent internalization of the virus, which remains infectious. The mechanism of viral transmission from DC to T cells is currently unknown. Sentinel immature DC (iDC) develop into Th1-promoting effector DC1 or Th2-promoting DC2, depending on the activation signals. We studied the ability of these effector DC subsets to support HIV-1 transmission in vitro. Compared with iDC, virus transmission is greatly upregulated for the DC1 subset, whereas DC2 cells are inactive. Increased transmission by DC1 correlates with increased expression of ICAM-1, and blocking studies confirm that ICAM-1 expression on DC is important for HIV transmission. The ICAM-1-LFA-1 interaction is known to be important for immunological cross talk between DC and T cells, and our results indicate that this cell-cell contact is exploited by HIV-1 for efficient transmission.

Triggering of innate immune responses by schistosome egg glycolipids and their carbohydrate epitope GalNAc beta 1-4(Fuc alpha 1-2Fuc alpha 1-3)GlcNAc.

To investigate the interactions of glycoconjugates with the innate immune system, peripheral blood mononuclear cells were stimulated with glycolipids derived from Schistosoma mansoni eggs and worms and with biochemically synthesized neoglycoconjugates. Egg glycolipids stimulated the production of interleukin (IL)--10, IL-6, and tumor necrosis factor--alpha in monocytes, whereas worm glycolipids failed to do so. When monoclonal antibodies that specifically recognize defined carbohydrate epitopes were used, the binding of a GalNAc beta 1-4(Fuc alpha 1-2Fuc alpha 1-3)GlcNAc (LDN-DF) reactive antibody was pronounced on egg glycolipids but was absent on worm glycolipids. The binding of antibodies that recognize Gal beta 1-4(Fuc alpha 1-3)GlcNAc (LewisX), GalNAc beta 1-4GlcNAc (LDN), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (LDN-F) was comparable for both preparations. Cytokine production in response to neoglycoconjugates containing enzymatically synthesized glycans also was measured. The LDN-DF neoglycoconjugate was the most potent cytokine inducer, which indicates that this difucosylated glycan can act at the host-parasite interface and can trigger innate immune responses.

Microbial compounds selectively induce Th1 cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse th cell-polarizing signals.

Upon microbial infection, specific Th1 or Th2 responses develop depending on the type of microbe. Here, we demonstrate that different microbial compounds polarize the maturation of human myeloid dendritic cells (DCs) into stably committed Th1 cell-promoting (DC1) or Th2 cell-promoting (DC2) effector DCs that polarize Th cells via different mechanisms. Protein extract derived from the helminth Schistosoma mansoni induced the development of DC2s that promote the development of Th2 cells via the enhanced expression of OX40 ligand. Likewise, toxin from the extracellular bacterium Vibrio cholerae induced development of DC2s as well, however, via an OX40 ligand-independent, still unknown mechanism. In contrast, toxin from the intracellular bacterium Bordetella pertussis induced the development of DC1s with enhanced IL-12 production, which promotes a Th1 cell development. Poly(I:C) (dsRNA, mimic for virus) induced the development of extremely potent Th1-inducing DC1, surprisingly, without an enhanced IL-12 production. The obtained DC1s and DC2s are genuine effector cells that stably express Th cell-polarizing factors and are unresponsive to further modulation. The data suggest that the molecular basis of Th1/Th2 polarization via DCs is unexpectedly diverse and is adapted to the nature of the microbial compounds.

Intercellular adhesion molecule-1/LFA-1 ligation favors human Th1 development.

Th cell polarization toward Th1 or Th2 cells is strongly driven by exogenous cytokines, in particular IL-12 or IL-4, if present during activation by Ag-presenting dendritic cells (DC). However, additional Th cell polarizing mechanisms are induced by the ligation of cell surface molecules on DC and naive Th cells. In the present study, the role of LFA-1/ICAM-1 ligation in human Th cell polarization was investigated. Triggering of LFA-1 on anti-CD3/CD28 stimulated naive Th cells with immobilized Fc-ICAM-1, in the absence of DC and exogenous cytokines, induced a marked shift toward Th1 cell development, accompanied by a dose-dependent decrease in GATA-3 expression and a dose-dependent increase in T-bet expression. Th1 polarization by LFA-1 ligation could be demonstrated only under low cytokine conditions, as it was largely overruled by IL-12 or IL-4. This IL-12-independent Th1-driving mechanism appears to be operated by certain subsets of effector DC. Maturation of DC by poly(I:C), a synthetic dsRNA, used as an in vitro model for viral infections, leads to the generation of Th1-driving effector DC (DC1), which express elevated levels of ICAM-1 but produce only low levels of IL-12p70. Blocking the ICAM-1/LFA-1 interaction in cocultures of these DC with naive Th cells attenuated their Th1-driving capacity. The molecular mechanism by which LFA-1 signaling supports Th1 differentiation is blocked by specific inhibitors of extracellular signal-regulated kinase phosphorylation. The present data indicate the existence of an IL-12-independent, extracellular signal-regulated kinase-mediated mechanism, through which high ICAM-1-expressing DC1 can drive Th1 polarization. This mechanism may be operational during viral infections.