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* Children’s Hospital, Albert-Ludwigs University, Freiburg, Germany;
Dipartimento di Patologia e Microbiologia Sperimentale, Università di Messina, Messina, Italy;
Channing Laboratory, Brigham and Women’s Hospital, and Departments of Medicine, Microbiology, and Molecular Genetics, Harvard Medical School, Boston, MA 02115; and
Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605
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Abstract |
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B kinase. Furthermore, JNK was essential for the transcriptional activation of inflammatory cytokine genes in response to GBS. Inhibition of JNK by the anthrapyrazolone SP600125 abrogated GBS-induced cytokine formation via an AP-1- and NF-B-dependent mechanism without impairing antibacterial properties such as phagocytosis of GBS and the formation of intracellular oxidative species. In contrast, inhibition of the MAPK p38 impaired both antibacterial processes. In a neonatal mouse model of GBS sepsis SP600125 inhibited the inflammatory response and improved survival. In conclusion, JNK plays a major role in the inflammatory, but not in the direct antibacterial response to inactivated GBS, and may thus serve as a rational target for an adjunctive GBS sepsis therapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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With respect to GBS, considerable evidence has been gathered that antibacterial properties are impaired in newborn infants, whereas immune cells from these infants are capable of mounting a powerful inflammatory response to GBS and other organisms (7, 8, 9). In a previous study, we found inactivated GBS to initiate a vigorous inflammatory cytokine response in phagocytes that by far exceeded the response to similar preparations of S. pneumoniae (10). The response to GBS was mediated by an as yet to be identified TLR or a functionally similar receptor and the TLR adapter molecule myeloid differentiation primary response protein (MyD88) (11). Furthermore, the MyD88-dependent pathway was not involved in phagocytosis of GBS; however, it critically determined the formation of antibacterial oxidant species such as peroxynitrate (10). Accordingly, MyD88 appears not to be an ideal target of therapeutic interventions in GBS sepsis because both the potentially detrimental proinflammatory activity and beneficial clearance would be impaired.
Downstream of MyD88, the family of at least five MAPKs orchestrates a host of phagocyte functions ranging from polymerization of the actin cytoskeleton to the phosphorylation of transcription factors resulting in the activation of inflammatory genes (12). In this study, we aimed first to establish the molecular basis for differences in GBS- and S. pneumoniae-induced formation of inflammatory cytokines and found the MAPK JNK to mediate the inflammatory effect of GBS by a transcriptional mechanism involving the transcription factors NF-B and AP-1. We then went on to use this knowledge to determine the effect of JNK inhibition on the phagocyte response to GBS in vitro and in vivo. We found JNK to be critical in the inflammatory, but not antibacterial responses to GBS. Hence, JNK appears to be an attractive molecular target of adjunctive GBS sepsis therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Reagents were obtained from Sigma-Aldrich, unless stated otherwise. PBS, DMEM, G418, and trypsin/versene mixture were purchased from Cambrex. Low endotoxin FBS was obtained from HyClone. LPS derived from Escherichia coli strain 0111:B4 was purchased from Sigma-Aldrich and twice re-extracted by phenol chloroform, as described. SP600125 was acquired from A.G. Scientific, and SB203580 was purchased from Calbiochem.
Generation of ethanol-inactivated GBS and S. pneumoniae
GBS type III strain COH1, initially isolated from a newborn infant with sepsis, and S. pneumoniae serotype-2 strain D39 have been described previously (13, 14, 15, 16). All strains were grown on blood agar plates (REMEL). Bacterial colonies were removed from the plates after overnight culture, washed three times in PBS, and then used to inoculate chemically defined medium prepared from endotoxin-free water (for GBS (17)) or DMEM enriched with 10% FBS (for S. pneumoniae) and grown to mid-log phase (adsorption650 = 0.27–0.30). Subsequently, bacteria were harvested by centrifugation and ethanol inactivated (70% ethanol, 45 min, on ice), washed once with water, and resuspended in pyrogen-free water at a concentration of 20 mg/ml (corresponding to 
1 x 1010 organisms/ml as determined by CFU/ml before inactivation). Accordingly, 200 µg/ml dry weight corresponds to 1 x 108 organisms/ml (as depicted in the figures). The entire procedure was performed under pyrogen-free conditions, resulting in preparations that were essentially free of endotoxin, as determined by a highly LPS-sensitive reporter system (Chinese hamster ovary-CD14 with E-selectin promoter-driven CD25, lower limit of detection 10–100 pg/ml).
Macrophages
Eight-week-old C57BL/6 mice (The Jackson Laboratory) were injected i.p. with 2.5 ml of a 3% thioglycolate solution (REMEL). After 72–96 h, peritoneal exudate cells were harvested by lavage with RPMI 1640 medium containing 10% FBS and 10 µg of ciprofloxacin/ml. The cells were washed with medium, counted in a hemocytometer, and plated. After 24–72 h, nonadherent cells were removed by washing with medium, and adherent cells were stimulated. RAW 264.7 mouse macrophages were cultured in DMEM containing 10% FBS and 10 µg of ciprofloxacin/ml.
Transfection of RAW 264.7 macrophages with reporter constructs
RAW 264.7 cells were seeded into 96-well tissue culture plates at a density of 105 cells/well. The following day, cells were transiently transfected with luciferase reporter constructs comprising minimal AP-1 and NF-B promoters (both from Stratagene) or human wild-type and mutant human TNF promoters (provided by N. Mackman, The Scripps Research Institute, La Jolla, CA) using Fugene (Roche), per the manufacturer’s recommendations. In individual experiments, cells were cotransfected with a constitutively active Renilla-luciferase reporter gene (Promega) to normalize between conditions for transfection efficacy. The following day, the cells were stimulated as indicated. After 4–6 h of stimulation, the cells were lysed in passive lysis buffer (Promega), and reporter gene activity was measured using a plate reader luminometer (MicroLumat Plus; Berthold Detection Systems). In all cases, the data shown represent one of at least three separate, but similar experiments, and are presented as the mean values ± SD of triplicate samples.
Measurement of proinflammatory activity
Nuclear translocation of AP-1 in primary mouse macrophages was determined exactly as described previously (10), with the only alteration that a 32P-labeled AP-1-specific oligonucleotide was used. For determination of TNF, RAW cells were seeded at a density of 1 x 105 cells/ml in 96-well dishes in DMEM with 10% FBS plus 10 µg of ciprofloxacin/ml and incubated over 16 h at 37°C in a 5% humidified CO2 environment. Supernatants were processed directly for the determination of released TNF by ELISA (R&D Systems), per the manufacturer’s protocols.
FITC labeling of GBS and determination of internalization
Ethanol-inactivated GBS (3 x 109/ml) were incubated with 0.3 mg of FITC/ml for 60 min on a rotating platform. FITC-labeled GBS were washed four times in PBS, and homogenous distribution of FITC labeling was confirmed by FACS analysis. Twenty-four-well dishes were plated with 2 x 105 RAW macrophages/well. RAW cells were incubated on the following day with FITC-labeled GBS. Following incubation, cells were washed, incubated for 60 s with 0.2% trypan blue (to quench extracellular fluorescence (18)), washed again, scraped into suspension with a rubber policeman, and fixed with 2% paraformaldehyde. The number of cells positive for FITC-GBS was determined by FACS.
Formation of peroxynitrate in mouse macrophages
RAW macrophages (2 x 105/well) were plated in 24-well tissue culture dishes. After 24 h, cells were incubated with the indicated concentrations of SB203580 or SP600125 for 30 min at 37°C. Then GBS and 0.15 µg of dihydrorhodamine 123/ml (Molecular Probes) were added, as indicated. After a 2-h incubation, the reaction was terminated by washing the cells with ice-cold PBS. The cells were detached with a rubber policeman, fixed with 2% paraformaldehyde, and analyzed by flow cytometry. The data shown represent one of three or more separate, but similar experiments, and are presented as the mean values ± SD of triplicate samples.
Neonatal mouse model of GBS sepsis
Neonatal (
24-h-old) BALB/c mice were used. Parental mice were obtained from Charles River Italia. Pups from each litter were randomly assigned to control or experimental groups, marked, and kept with the mother. GBS type III strain COH1 was grown to the mid-log phase in Todd-Hewitt broth (Oxoid) and were diluted to the appropriate concentration in PBS (0.01 M phosphate and 0.15 M NaCl (pH 7.2)). Then mouse pups were injected s.c. (30 µl) with GBS dosages, as indicated. In each experiment, the number of injected bacteria was determined by colony counts on blood agar (Oxoid). SP600125 or vehicle (40% polyethylene glycol 400 in PBS) was administered s.c. 3 h before challenge. Mice were observed daily for 6 days after infection. In this model, deaths are rarely observed after 5 days. In addition, some of the mice were killed at the indicated time points to measure bacterial burden and plasma TNF levels.
All of the procedures were in agreement with the guidelines of the European Commission for the handling of laboratory animals, and the studies presented in this work were approved by the relevant national and institutional committees.
Analysis of TNF concentrations and bacterial burden in neonatal mice
Neonatal mice were killed by decapitation under anesthesia at the indicated times after GBS challenge. Mixed venous-arterial blood was collected in heparinized containers and centrifuged after 10 µl was saved for colony counts. For CFU measurement, blood (10 µl) was diluted 1/20 in PBS, whereas kidneys and spleens were homogenized in PBS. Serial dilutions were prepared in duplicates and plated on blood agar. Bacterial CFU were assessed after overnight growth at 37°C.
TNF concentrations were determined in duplicates in pooled plasma from four animals using a commercial ELISA kit (mouse TNF module set; Bender MedSystems). The lower detection limit of this assay was 16 pg/ml.
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Results |
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Inactivated GBS is a potent stimulus for macrophages via a MyD88-dependent mechanism that appears to be at least partially distinct from activation by S. pneumoniae. GBS is much more potent in inducing TNF and IL-6 than other virulent streptococcal species, i.e., S. pneumoniae (Fig. 1, A and B). This phenomenon is not only restricted to mouse macrophages; we found similar results using human PBMC (data not shown). Similar results were obtained by using other bacterial strains, all of which are highly virulent in mice (NEM 316 for GBS, and WU2 for S. pneumoniae) (13, 19). Therefore, we determined whether the differences found in TNF protein formation were due to different transcriptional activation by GBS and S. pneumoniae. We used a reporter construct that comprises the TNF promoter linked to the luciferase gene. In this study, we found substantially different transcriptional activation by these two streptococcal species corresponding to the data on protein formation (Fig. 1C).
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B kinase activation and differ slightly in NF-B activation
Next, we attempted to elucidate the molecular basis of the differential activation of the TNF promoter by GBS and S. pneumoniae. NF-B has been described as a critical transcription factor for GBS-induced TNF (10, 20). In this study, we aimed to define a distinct NF-B binding site in the TNF promoter that is essential for GBS-induced TNF formation. To this end, we used a TNF reporter construct that contained a mutation in the NF-B3 site of the TNF promoter. Whereas GBS robustly activated the TNF reporter comprising the wild-type promoter sequence, this activation was essentially abrogated when the NF-B3 site was mutated, confirming the dependency of GBS-induced TNF activation on the NF-B3 binding site (Fig. 2A). In contrast to TNF, IB kinase, which leads via IB degradation to NF-B liberation and translocation to the nucleus, was similarly induced by both GBS and S. pneumoniae (Fig. 2B). Then we compared the ability of GBS and S. pneumoniae to activate a reporter that contains solely six copies of the NF-B consensus sequence as the promoter. Surprisingly, and in contrast to NF-B liberation, GBS induced activation of NF-B more potently than S. pneumoniae (Fig. 2C). These findings are of importance because they indicated that the stimulation with inactivated S. pneumoniae potently activates signaling intermediates (IB kinase) in macrophages, although they poorly induce the formation of inflammatory cytokines. Second, these results imply that GBS and S. pneumoniae differ in their ability to induce the NF-B pathway downstream of IB kinase, because transcriptional NF-B activation, but not IB kinase activation, differed between these bacterial species.
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AP-1-restricted transcriptional activation of TNF
Next to NF-B, AP-1 partially accounts for TNF transcriptional activation in response to diverse microbial structures (21). Therefore, we analyzed the role of the AP-1 site in the TNF promoter in TNF promoter activation by GBS. We found that mutations in the TNF promoter that were shown previously to correspond to the AP-1 site (–65 T-G, –66 G-T) abolish activation of the TNF promoter by GBS, indicating that binding of transcription factors to AP-1 is indeed a prerequisite for TNF induction. In contrast, mutations in the AP-2 site (–34 C-T, –31 G-T) that lies adjacent to the AP-1 site within the TNF promoter remained essentially without effect on transcriptional activation of the TNF promoter by GBS (Fig. 3A). Furthermore, GBS exceeded S. pneumoniae with respect to the induction of nuclear translocation and DNA binding of AP-1 transcription factors (Fig. 3B) and stimulation of a minimal AP-1 reporter (Fig. 3C). Two transcription factor heteromers have been shown previously to bind to the AP-1 site, and both involve the transcription factor c-Jun (c-Jun/c-Phos, c-Jun/activating transcription factor 2 (ATF2)). c-Jun needs to be posttranscriptionally phosphorylated at the residues Ser63 and Ser73 by interaction of JNK with the c-Jun NH2-terminal activation domain to bind to the AP-1 site (22). Accordingly, we tested whether GBS and S. pneumoniae differed in inducible activity of JNK, thus resulting in different AP-1 activity. Phosphorylation of the JNK substrate c-Jun can be monitored by specific Abs. In concordance with the data on AP-1 activation, GBS clearly exceeded S. pneumoniae in the activation of the JNK (Fig. 3D).
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We subsequently tested whether activation of c-Jun mediated the TNF response to GBS. We found that c-Jun phosphorylation in macrophages was completely inhibited by preincubation with the anthrapyrazolone SP600125, a known inhibitor of JNK (Fig. 4A). In additional experiments, we found that JNK was critical for GBS-induced TNF formation. SP600125 at concentrations as low as 10 µM abrogated the TNF response (Fig. 4B). This inhibitory effect occurred on a (pre)transcriptional level, because, similar to TNF protein, SP600125 inhibited TNF promoter activation by GBS in a dose-dependent fashion (Fig. 4C). Similar to TNF, formation of IL-6 by mouse macrophages (RAW 264.7) was inhibited by SP600125 (data not shown). This indicates that JNK and AP-1 are critical for GBS-induced cytokine induction beyond TNF.
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In this study, we addressed whether SP600125-mediated inhibition of GBS-induced JNK and TNF transcription was correlated with inhibition of AP-1. Indeed, we found that SP600125 inhibited GBS-induced AP-1 activation (Fig. 5A). Clearly, considerable cross talk occurs between the signaling pathways that comprise the MAPKs p38 and JNK both among upstream MAPK kinases and downstream transcription factors such as AP-1. Thus, we determined the effect of SP600125 on p38 activation in response to GBS. As previously reported, p38 is rapidly phosphorylated in GBS-stimulated mouse macrophages (10, 20). Interestingly, this activation was not inhibited by SP600125; instead, we observed some increased activation when the macrophages were incubated in the presence of GBS and SP600125 as compared with GBS alone (Fig. 5B). This finding has two important implications: first, JNK appears to be absolutely required for GBS-induced AP-1 (and TNF) activation, and p38 cannot compensate for JNK inhibition. Second, SP600125 appears to be a specific and potent inhibitor of GBS-activated JNK.
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B transactivation without impairing IB degradation
To further explore the effect of JNK inhibition on the phagocyte response to GBS, we analyzed NF-B in this context. Interestingly, IB degradation as the first step in NF-B activation was not impaired by SP600125, indicating that it did not interfere with activity of IB kinase (Fig. 6A). However, NF-B transactivation was affected because SP600125 inhibited activity of a reporter that comprises a minimal promoter with six NF-B binding sites only (Fig. 6B). Accordingly, JNK is directly involved and essential in the transactivation of NF-B in response to GBS. To summarize these findings, JNK is an essential signaling intermediate in both the AP-1- and NF-B-dependent signaling pathways engaged by GBS, therefore occupying a central position in the activation of TNF.
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MAPKs have been implicated previously in antibacterial properties of macrophages, in particular uptake of bacteria (23). Hence, we addressed whether JNK was involved in this process in response to GBS. Incubation of macrophages with FITC-labeled ethanol-inactivated GBS resulted in rapid uptake of GBS irrespective of the presence of SP600125. In sharp contrast, SB203580, a pyridinyl imidazole-based inhibitor of the MAPK p38, abrogated GBS uptake in a dose-dependent fashion (Fig. 7 and data not shown).
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B (10). Similar to the effects observed with respect to phagocytosis inhibition of p38 by SB203580, but not inhibition of JNK, impaired peroxynitrate formation. In contrast, high concentrations of SP600125 resulted in constitutive up-regulation of peroxynitrate, probably due to some compensatory induction of p38 (Fig. 8).
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Because SP600125 inhibited GBS-induced inflammatory responses in vitro without impairing other phagocyte properties, we wondered whether this compound might affect survival of mice suffering from GBS sepsis. To this end, we used a s.c. mouse model that mimics GBS septic shock in newborn infants (LD90). In this model, three key findings were made: first, SP600125 improved the outcome in neonatal sepsis in a dose-dependent fashion (Fig. 9A). Using Kaplan-Meier statistics, SP600125 significantly improved survival at 2 mg/kg as compared with vehicle control (p = 0.0005). Further statistical values (Kaplan-Meier) were p = 0.068 for SP600125 at 0.2 mg/kg and p = 0.67 for 0.05 mg/kg. Notably, no further deaths were observed after day 3. Second, SP600125 impaired the TNF response to GBS. Both at 12 and 24 h, TNF blood levels were higher in vehicle controls than in animals treated with at least 0.2 mg of SP600125/mouse (Fig. 9B). Finally, SP600125 did not affect bacterial clearance, neither in circulating blood nor in liver or spleen (Fig. 9C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B, which are critical for activation of the TNF promoter. JNK protein kinases are encoded by three genes with tissue-specific expression profiles. Whereas Jnk1 and Jnk2 genes are ubiquitously expressed, Jnk3 seems to be largely restricted to brain, heart, and testis. Through alternative splicing, 10 JNK isoforms are created. Accordingly, extensive complementation occurs between the Jnk genes in mice with targeted deletions of individual JNK genes. Furthermore, targeted deletion of the common upstream MAPK kinases 4 and 7 is lethal in mice. Hence, compound mutants that lack expression of all JNK isoforms are required to examine a JNK-deficient phenotype (30).
Alternatively, somewhat broader inhibitors of all three JNK isoforms can be used. The chemical inhibitor tested in this study was the anthrapyrazolone SP600125, a small molecule that reversibly inhibits JNK with a >20-fold selectivity as compared with other enzymes (31). In our hands, inhibition of JNK (by SP600125) completely abrogated the GBS-induced TNF formation. This finding seemed particularly important in the context with GBS because TNF is a critical mediator of lethality in a neonatal mouse model of GBS sepsis (32). JNK activity probably affects TNF formation by both transcriptional and posttranscriptional mechanisms. First, it induces transcription factor binding to the TNF promoter. Second, it appears to promote stability of TNF mRNA (31). The striking phenotype of the MyD88-deficient mouse with respect to GBS-induced TNF formation (11, 32) might be in part due to a significant drop in mRNA stability upon ligation of TLRs.
It is well established that beyond the principal target c-Jun, JNK also phosphorylates Ser/Thr-Pro motifs in other AP-1-binding transcription factors, including JunB, JunD, and ATF2 (33). JNK and its phosphorylation target participate in complex regulatory processes of AP-1 activity, such as modulating the transcriptional coactivator CBP/p300, histone acetylation by ATF2, and ubiquitin-mediated degradation of AP-1-binding proteins (30). Accordingly, even though SP600125 inhibited JNK with considerable specificity, collateral effects beyond c-Jun have to be considered. Indeed, we consistently found some up-regulation of IB degradation, p38 phosphorylation, and formation of peroxynitrate as a consequence of coincubation with GBS plus SP600125. However, these events did not affect the formation of inflammatory cytokines, because inhibition of JNK resulted in abrogation of the inflammatory cytokine response irrespective of the effect on p38 and IB. Furthermore, these compensatory effects were modest and only occurred at SP600125 concentrations clearly exceeding those needed to decrease TNF formation.
It seems noteworthy that the inflammatory phenotype of macrophages incubated with GBS plus SP600125 mimics the activation by S. pneumoniae alone (potent NF-B liberation, but impaired transactivation of NF-B and AP-1 and abrogated TNF formation). Hence, it is tempting to speculate that differences in TNF formation induced by these two streptococcal species are due to differences in JNK activation.
Adjunctive sepsis therapy that targets proinflammation in the early stages of disease (before the host immune response is in part shut down) ideally has to involve a potent inhibition of inflammatory mediators while conserving directly antibacterial properties of phagocytes (26). Inhibition of JNK did not interfere with phagocytosis or the formation of antibacterial oxygen species. In contrast, inhibition of p38, a previously suggested target in antisepsis therapy, impairs both antibacterial properties.
Despite largely independent control mechanisms of inflammatory cytokine formation and bacterial clearance, both properties are most likely linked in vivo. As an example, TNF Abs impair the clearance of a low inoculum of GBS in a sepsis model (32) most likely due to impaired priming of phagocytes. Accordingly, it seemed essential to carefully assess the role of JNK and the effect of its inhibition in an in vivo sepsis model. Similar to what we observed, in vitro inhibition of JNK abrogated the TNF response of GBS-infected neonatal mice without a significant effect on bacterial clearance. Moreover, inhibition of JNK clearly improved survival in this sepsis model despite the high bacterial load.
Several issues need to be considered to further evaluate JNK as a therapeutic target in neonatal GBS sepsis. First, whereas inhibition of TNF formation is beneficial in septic shock both in rodents and in humans (32), this inhibition may interfere with proper bacterial clearance in early stages of invasive GBS disease. Second, it is currently unknown at what point of time within the course of sepsis JNK inhibition is most effective and when it no longer influences the outcome. These studies are underway. Despite these important unresolved questions, to our best knowledge this study represents the first successful use of a JNK inhibitor in the modulation of Gram-positive sepsis in vivo.
In conclusion, JNK specifically mediates the inflammatory cytokine response in GBS septic shock. Inhibition of JNK improves outcome in GBS-mediated sepsis without interfering with antibacterial phagocyte functions both in vitro and in vivo. Thus, JNK constitutes a rational target for adjunctive therapy of neonatal GBS sepsis.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by Deutsche Forschungsgemeinschaft Grant He 3127/2-1 (to P.H.) and by the National Institutes of Health Grants AI52455 and GM54060 (to D.T.G.). 
2 S.K. and G.M. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Philipp Henneke, Children’s Hospital, Albert-Ludwigs University, Mathildenstr.1, 79106 Freiburg, Germany. E-mail address: henneke{at}kikli.ukl.uni-freiburg.de
4 Abbreviations used in this paper: GBS, group B streptococcus; ATF2, activating transcription factor 2.
Received for publication September 23, 2005. Accepted for publication December 28, 2005.
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References |
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B are involved in TNF-
responses to group B streptococci. J. Immunol. 169: 1401-1409. promoter in human monocytic cells: regulation by Egr-1, c-Jun, and NF-B transcription factors. J. Biol. Chem. 272: 17795-17801. B activation and TNF production induced by lipopolysaccharide and group B streptococcal cell walls. J. Immunol. 160: 4535-4542. gene expression and activation of the transcription factors NF-B and activator protein-1 in human cord blood monocytes. J. Immunol. 165: 419-425. This article has been cited by other articles:
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  S. Kenzel, S. Santos-Sierra, S. D. Deshmukh, I. Moeller, B. Ergin, K. A. Fitzgerald, E. Lien, S. Akira, D. T. Golenbock, and P. Henneke Role of p38 and Early Growth Response Factor 1 in the Macrophage Response to Group B Streptococcus Infect. Immun., June 1, 2009; 77(6): 2474 - 2481. [Abstract] [Full Text] [PDF]
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 I. Venza, M. Cucinotta, M. Visalli, G. De Grazia, S. Oliva, and D. Teti Pseudomonas aeruginosa Induces Interleukin-8 (IL-8) Gene Expression in Human Conjunctiva through the Recruitment of Both RelA and CCAAT/Enhancer-binding Protein {beta} to the IL-8 Promoter J. Biol. Chem., February 13, 2009; 284(7): 4191 - 4199. [Abstract] [Full Text] [PDF]
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 G. Adhikary, Y. Sun, and E. Pearlman C-Jun NH2 terminal kinase (JNK) is an essential mediator of Toll-like receptor 2-induced corneal inflammation J. Leukoc. Biol., April 1, 2008; 83(4): 991 - 997. [Abstract] [Full Text] [PDF]
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 G. Mancuso, A. Midiri, C. Biondo, C. Beninati, S. Zummo, R. Galbo, F. Tomasello, M. Gambuzza, G. Macri, A. Ruggeri, et al. Type I IFN Signaling Is Crucial for Host Resistance against Different Species of Pathogenic Bacteria J. Immunol., March 1, 2007; 178(5): 3126 - 3133. [Abstract] [Full Text] [PDF]
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 I. Venza, M. Cucinotta, S. Caristi, G. Mancuso, and D. Teti Transcriptional Regulation of IL-8 by Staphylococcus aureus in Human Conjunctival Cells Involves Activation of AP-1 Invest. Ophthalmol. Vis. Sci., January 1, 2007; 48(1): 270 - 276. [Abstract] [Full Text] [PDF]
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 M. A. Bogoyevitch and B. Kobe Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 1061 - 1095. [Abstract] [Full Text] [PDF]
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) or a TNF.luc reporter plasmid with a mutation in the 
). The next day, cells were stimulated with inactivated GBS over 5 h, and reporter activity was determined by luminometry. B, RAW 264.7 cells were stimulated with GBS or S. pneumoniae (10x/ml) over 20 min. Then cellular lysates were subjected to Western blot analysis with I
) and stimulated and analyzed by luminometry, as depicted in Fig. 1
