Interleukin-6 RNA Research Articles

Dual analysis of IL-8 in non-small cell lung cancer: A clinical perspective on protein and RNA evaluation via imaging mass cytometry.

e15068 Background: The cytokine Interleukin-8 (IL-8) plays a pivotal role in the tumorigenesis and progression of non-small cell lung cancer (NSCLC), influencing both the tumor microenvironment and immune escape mechanisms. Given its significance, accurately assessing IL-8 levels at the protein and RNA levels could provide valuable insights into its prognostic value and inform therapeutic targeting strategies. This study utilizes imaging mass cytometry (IMC) to conduct a comprehensive evaluation of IL-8 expression in NSCLC, integrating both protein presence and gene expression analysis within the spatial context of the tumor microenvironment. Methods: Our approach leverages the capabilities of IMC to simultaneously detect protein and RNA levels of IL-8 in NSCLC tissue samples. For protein analysis, antibodies tagged with heavy metal isotopes specifically target IL-8, allowing for precise quantification and spatial localization within the tumor. Concurrently, RNA detection is achieved through hybridization probes designed for IL-8 mRNA, enabling the direct observation of gene expression patterns. This method provides a unique opportunity to correlate IL-8's protein abundance with its mRNA expression, offering insights into the regulatory mechanisms at play and their impact on the tumor's behavior and patient outcomes. Results: Preliminary data demonstrate a marked heterogeneity in IL-8 distribution at both the protein and RNA levels, with distinct patterns emerging that relate to tumor aggressiveness and immune infiltration. High IL-8 expression, identified through our integrated IMC approach, correlates with poor prognosis and reduced response to standard therapies, underscoring its potential as a biomarker for NSCLC management. Furthermore, the spatial analysis afforded by IMC highlights the microenvironmental niches where IL-8-mediated interactions could drive tumor progression and immune evasion. Conclusions: By employing IMC for the dual analysis of IL-8 protein and RNA in NSCLC, our study provides a comprehensive view of this cytokine's role in lung cancer pathology. The ability to visualize and quantify IL-8 expression within the spatial context of the tumor microenvironment opens new avenues for understanding its biological impact and for developing targeted therapies. Our findings advocate for the inclusion of IL-8 in the biomarker panel for NSCLC, with potential implications for patient stratification and personalized treatment planning. Future research will focus on expanding these observations to a larger cohort and exploring the therapeutic modulation of IL-8 as a strategy for improving NSCLC outcomes.

Elevated interleukin-8 expression by skin fibroblasts as a potential contributor to pain in women with Fabry disease.

Fabry disease (FD) is a lysosomal storage disorder of X-linked inheritance. Mutations in the α-galactosidase A gene lead to cellular globotriaosylceramide (Gb3) depositions and triggerable acral burning pain in both sexes as an early FD symptom of unknown pathophysiology. We aimed at elucidating the link between skin cells and nociceptor sensitization contributing to FD pain in a sex-associated manner. We used cultured keratinocytes and fibroblasts of 27 adult FD patients and 20 healthy controls. Epidermal keratinocytes and dermal fibroblasts were cultured and immunoreacted to evaluate Gb3 load. Gene expression analysis of pain-related ion channels and pro-inflammatory cytokines was performed in dermal fibroblasts. We further investigated electrophysiological properties of induced pluripotent stem cell (iPSC) derived sensory-like neurons of a man with FD and a healthy man and incubated the cells with interleukin 8 (IL-8) or fibroblast supernatant as an in vitro model system. Keratinocytes displayed no intracellular, but membrane-bound Gb3 deposits. In contrast, fibroblasts showed intracellular Gb3 and revealed higher gene expression of potassium intermediate/small conductance calcium-activated potassium channel 3.1 (KCa 3.1, KCNN4) in both, men and women with FD compared to controls. Additionally, cytokine expression analysis showed increased IL-8 RNA levels only in female FD fibroblasts. Patch-clamp studies revealed reduced rheobase currents for both iPSC neuron cell lines incubated with IL-8 or fibroblast supernatant of women with FD. We conclude that Gb3 deposition in female FD patient skin fibroblasts may lead to increased KCa3.1 activity and IL-8 secretion. This may result in cutaneous nociceptor sensitization as a potential mechanism contributing to a sex-associated FD pain phenotype.

Conspectus on nanodiagnostics as an incipient platform for detection of oral potentially malignant disorders and oral squamous cell carcinoma.

Conspectus on nanodiagnostics as an incipient platform for detection of oral potentially malignant disorders and oral squamous cell carcinoma.

Iron, hepcidin, and microcytosis in canine hepatocellular carcinoma.

Erythrocyte microcytosis in some dogs with hepatocellular carcinoma (HCC) suggests a derangement in systemic iron. Hepcidin, the master regulator of iron, is secreted by the liver in response to interleukin 6 (IL-6) and/or bone morphogenetic protein 6 (BMP6) and can cause microcytosis. Pilot study to compare the quantities of hepcidin, IL-6, and BMP6 RNA molecules in archival tumoral (HCC) and adjacent peritumoral (non-HCC) hepatic tissue to determine if they are different between tissue types or associated with microcytosis. RNA was isolated from formalin-fixed, paraffin-embedded HCC and non-HCC tissue from seven microcytic dogs and four normocytic dogs. Digital RNA counts of hepcidin, IL-6, or BMP6, and six other iron-regulatory genes were determined using the Nanostring nCounter system. The area of blue on each section was digitally evaluated to measure the extent of Prussian blue staining objectively. Parameters were compared between HCC and non-HCC tissue and between microcytic and normocytic groups. Hepcidin was decreased, and transferrin receptor 1 (TfR1) was increased in HCC tissue compared with non-HCC tissue. Non-HCC hepcidin RNA counts correlated negatively with MCV and positively with the extent of iron staining. Hepcidin expression was higher in non-HCC tissue of microcytic cases than in normocytic cases. Canine HCC cases showed relatively increased iron staining in non-HCC tissue and decreased hepcidin RNA in HCC tissue. Microcytic cases had higher hepcidin RNA in non-HCC tissue than normocytic cases. Future studies may extend these findings to protein quantification, cellular localization of RNA changes, and determining if iron loading in canine liver is a predisposing factor for HCC.

Highlights From the Circulation Family of Journals.

Highlights From the Circulation Family of Journals.

Interleukin 6 Is Not Made By Chimeric Antigen Receptor T Cells and Does Not Impact Their Function

Introduction:Anti-CD19 chimeric antigen receptor T cells (CART19) generate unprecedented complete response rates of up to 90% in relapsing/refractory acute lymphoblastic leukemia. Associated with this therapy is a multi-symptom toxicity known as cytokine release syndrome (CRS). Clinically CRS resembles macrophage activation syndrome (MAS), with a pattern of cytokines in serum coupled with other biomarkers (such as ferritin) and physical findings (fever, sudden organomegaly, confusion). Clinical interest has focused on interleukin-6 (IL-6), as blocking this pathway with tocilizumab (an IL-6R antagonist) has relieved the most life-threatening aspects of CRS in patients. Nothing is known, however, about the mechanism behind the triggering of CAR associated CRS/MAS nor the cellular sources of the toxic cytokines. This is a critical lack of knowledge, as each CAR product may result in a different CRS resulting in different clinical outcomes and management strategies. We sought to identify the cellular source of IL-6 and other MAS cytokines specifically during a 41BB CAR mediated CRS, and cross validate with investigations into patient peripheral blood PBMCs during CRS.Results: Using a xenograft model of a primary patient leukemia and CART19 from a patient that experienced Grade 4 CRS, we measured cytokine production in the serum of animals 3 and 7 days post CART19. While we could detect GMCSF, IL-2 and IFNg easily, IL-6 was not detected and the animals did not appear ill during this phase despite disease response. Given the clinical similarities of CRS to MAS, we performed co-culture of CART19 T cells, Nalm-6 leukemia and cells derived in vitro from peripheral blood monocytes (which were autologous to the CAR T cells) including immature dendritic cells (iDC), mature dendritic cells (mDC) and macrophages. Similar to the in vivo results, coculture of CART19 and Nalm-6 produced high levels of GMCSF, IFNg, IL-2 and IL-10 but no detectable IL-6 or IL-8. Only in the presence of the monocyte lineage antigen presenting cells (APCs) did we observe IL-6 and IL-8 release (more than 100 fold increase over controls). Transwell in vitro experiments separating CART19/Nalm-6 from the APCs showed the same pattern, indicating the CART19 mediated killing of target cells induces the IL-6 release from APCs in a contact independent manner. Nanostring RNA analysis of separated cell populations indicated that IL-6 and IL-8 are exclusively produced by APCs, not CART19 or Nalm-6 (Figure 1). Both CD107a degranulation and the total Nanostring RNA profile of CART19 was not different in the presence or absence of APCs, indicating that an MAS-like CRS is likely not part of CART19 efficacy. Finally, we analyzed the peripheral blood mononuclear cells of 18 patients receiving CART19 for pediatric ALL by Nanostring. Patients with Grade 4 CRS and only circulating T cells showed no IL-6 or IL-8 RNA, confirming in vivo that CART19 cells are not the cellular source of IL-6 during CRS. Unsupervised clustering of the Nanostring profiles also revealed four distinct gene signatures: one for patients with only circulating leukemic blasts, two for Grade 2-3 CRS that clustered separately and one for Grade 4 CRS (Figure 2).Conclusions: Here we demonstrate that IL-6 as part of CRS is produced by APCs and not T cells in response to CART19 mediated killing of leukemia, and that CART19 cells do not seem affected by the presence of CRS cytokines either in transcriptional profile or killing potential. This data provides the rationale for blocking this toxic cytokine before symptoms appear without changing CART19 efficacy, in addition to supporting a rapid Nanostring based profile to identify prospectively the patients at risk for Grade 4 CRS. [Display omitted] [Display omitted] DisclosuresBarrett:Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy.

Hyperproduction of IL-6 caused by aberrant TDP-43 overexpression in high-fat diet-induced obese mice

Inclusion of Tat-activating regulatory DNA-binding protein-43 (TDP-43) due to hyperphosphorylation or hyperubiquitination is a cause of neurodegenerative disease. Cellular TDP-43 expression is tightly controlled through a negative feedback loop involving its mRNA. Recently, we reported that the TDP-43-mediated sub-nuclear body is an essential site of interleukin-6 (IL-6) pre-mRNA processing. Here we show that mice fed on a high-fat diet exhibit increased TDP-43 expression in the liver and adipose tissue with a prominent increase in IL-6. TDP-43 depletion in vivo reduces IL-6 production in the liver. Overexpression or depletion of TDP-43 in pre-adipose and adipose cells causes reciprocal alteration of IL-6 expression and RNA processing. Our findings provide evidence for a link between homeostasis of TDP-43 expression and the risk of developing obesity.

An animal model for chorioamnionitis at term

The purpose of this study was to develop an animal model for intrapartum inflammation at term to investigate the interactions between maternal and fetal inflammatory responses and adverse neurologic outcome. Lipopolysaccharide (160, 320, or 640 μg/kg) was administered intraperitoneally to day 20 term-pregnant Sprague Dawley rat dams 2, 4, and 6 hours before sample collection. Maternal outcomes included dam core temperature and plasma interleukin 6 (IL-6). Fetal outcomes included plasma IL-6, brain IL-6 messenger RNA expression, and brain IL-6 protein expression. Primary cortical cell cultures were prepared to examine neuronal morphologic condition. Neurite counts were obtained with the use of automated Sholl analysis. Maternal plasma IL-6 levels peaked 2 hours after lipopolysaccharide stimulus and rapidly resolved, except for an observed low level persistence at 6 hours with 640 μg/kg. Fetal plasma and placental IL-6 expression also peaked rapidly but only persisted in placental samples. Fetal brain IL-6 RNA and protein expression was significantly higher than control litters at 6 hours after the exposure to both 320 μg/kg (P ≤ .05) and 640 μg/kg (P ≤ .01). Cortical cells from fetuses that were exposed for 6 hours to maternal systemic inflammation showed reduced neurite number and neurite length (P < .001) with increasing lipopolysaccharide dose. Our results demonstrate that fetal brain injury follows isolated systemic maternal inflammation and that fetal brain inflammation lags after maternal stimulus, which creates a potential 4-hour clinical window for therapeutic intervention.

Urotensin II Induces Interleukin 8 Expression in Human Umbilical Vein Endothelial Cells

BackgroundUrotensin II (U-II), an 11-amino acid peptide, exerts a wide range of actions in cardiovascular systems. Interleukin-8 (IL-8) is secreted by endothelial cells, thereby enhancing endothelial cell survival, proliferation, and angiogenesis. However, the interrelationship between U-II and IL-8 as well as the detailed intracellular mechanism of U-II in vascular endothelial cells remain unclear. The aim of this study was to investigate the effect of U-II on IL-8 expression and to explore its intracellular mechanism in human umbilical vein endothelial cells.Methods/Principal FindingsPrimary human umbilical vein endothelial cells were used. Expression of IL-8 was determined by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and luciferase reporter assay. Western blot analyses and experiments with specific inhibitors were performed to reveal the downstream signaling pathways as concerned. U-II increased the mRNA/protein levels of IL-8 in human umbilical vein endothelial cells. The U-II effects were significantly inhibited by its receptor antagonist [Orn5]-URP. Western blot analyses and experiments with specific inhibitors indicated the involvement of phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase in U-II-induced IL-8 expression. Luciferase reporter assay further revealed that U-II induces the transcriptional activity of IL-8. The site-directed mutagenesis indicated that the mutation of AP-1 and NF-kB binding sites reduced U-II-increased IL-8 promoter activities. Proliferation of human umbilical vein endothelial cells induced by U-II could be inhibited significantly by IL-8 RNA interference.Conclusion/SignificanceThe results show that U-II induces IL-8 expression in human umbilical vein endothelial cells via p38 mitogen-activated protein kinase and extracellular signal-regulated kinase signaling pathways and IL-8 is involved in the U-II-induced proliferation of human umbilical vein endothelial cells.

IL-6 pathway-driven investigation of response to IL-6 receptor inhibition in rheumatoid arthritis

ObjectivesTo determine whether heterogeneity in interleukin-6 (IL-6), IL-6 receptor and other components of the IL-6 signalling pathway/network, at the gene, transcript and protein levels, correlate with disease activity in patients...

Markers of Intestinal Inflammation, Not Bacterial Burden, Correlate With Clinical Outcomes in Clostridium difficile Infection

Clostridium difficile is a leading hospital-acquired infection. Many patients remain symptomatic for several days on appropriate antibiotic therapy. To assess the contribution of ongoing infection vs persistent inflammation, we examined the correlation between fecal cytokine levels, fecal C. difficile burden, and disease outcomes in C. difficile infection (CDI). We conducted a prospective cohort study in Barnes Jewish Hospital between June 2011 and May 2012 of hospitalized adults with CDI. We determined fecal interleukin 8 (IL-8) and lactoferrin protein concentrations by enzyme immunoassay. We used real-time polymerase chain reaction (PCR) to measure relative fecal IL-8 and CXCL-5 RNA transcript abundances, and quantitative PCR to enumerate C. difficile burden. Of 120 study subjects, 101 (84%) were started on metronidazole, and 33 of those (33%) were subsequently given vancomycin. Sixty-two (52%) patients had diarrhea persistent for 5 or more days after starting CDI therapy. Initial fecal CXCL-5 messenger RNA (mRNA), IL-8 mRNA, and IL-8 protein correlated with persistent diarrhea and use of vancomycin. Time to diarrhea resolution was longer in patients with elevated fecal cytokines at diagnosis. Fecal cytokines were more sensitive than clinical severity scores in identifying patients at risk of treatment failure. Clostridium difficile burden did not correlate with any measure of illness or outcome at any point, and decreased equally with metronidazole and vancomycin. Persistent diarrhea in CDI correlates with intestinal inflammation and not fecal pathogen burden. These findings suggest that modulation of host response, rather than adjustments to antimicrobial regimens, might be a more effective approach to patients with unremitting disease.

An RNA Element in Human Interleukin 6 Confers Escape from Degradation by the Gammaherpesvirus SOX Protein

Several viruses express factors to silence host gene expression via widespread mRNA degradation. This phenotype is the result of the coordinated activity of the viral endonuclease SOX and the cellular RNA degradation enzyme Xrn1 during lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection. While most cellular transcripts are highly downregulated, a subset of host mRNA escapes turnover via unknown mechanisms. One of the most prominent escapees is the interleukin 6 (IL-6) mRNA, which accumulates robustly during KSHV lytic infection and is not subjected to SOX-induced degradation. Here we reveal that the IL-6 mRNA contains a dominant, cis-acting ∼100-nucleotide element within its 3' untranslated region (UTR) that renders it directly refractory to cleavage by SOX. This element specifically interacts with a cellular protein complex both in SOX-transfected cells and in KSHV-infected B cells. Using a directed RNA pulldown approach, we identified two components of this complex to be the AU-rich element (ARE) binding proteins AUF1 and HuR. Depletion of these proteins significantly reduced the protective capacity of the IL-6 RNA element in SOX-expressing cells. These findings suggest that SOX activity may be directly counteracted by select RNA regulatory complexes and reveal a novel mechanism contributing to the robust expression of IL-6 during KSHV replication.

Mycoplasma penetrans infection is a potential cause of immunoglobulin A nephropathy: a new animal model

A new animal model of immunoglobulin A nephropathy (IgAN) was made by infecting mice with Mycoplasma penetrans (Mpe). To examine the pathogenesis of IgAN induced by Mpe infection, tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and nuclear factor-kB (NF-kB) protein levels were compared among study groups. To make an experimental IgAN animal model, mice were infected with Mpe, SP-4 medium or phosphate-buffered saline (PBS) via the urinary tract. To compare changes in the classical IgAN model, TNF-alpha and IL-6 RNA expression levels were measured using RT-PCR, and NF-kB protein was measured using EMSA. By producing a urinary tract infection with Mpe, we developed a new animal model of IgAN with a 100% success rate. There was no difference with the classical animal model. We also observed IgG deposition in 66.67% of the Mpe-infection group. Glomerular cell and mesangial matrix proliferation was greater in the Mpe-infection group than in the control groups (p<0.05). In the Mpe-infection and classical groups, TNF-alpha and IL-6 expression levels were much higher than in the control groups (p<0.01). NF-kB expression was much higher in the Mpe-infection group (p<0.05). We made a new IgAN animal model that will offer a new direction for IgAN research. The activation of inflammation factors was associated with the Mpe induction of IgAN.

Interleukin-6 and its receptor in the development of in vitro fertilized ovine embryos

To study the effects of interleukin-6 (IL-6) and its receptor (IL-6R) at each stage in the early development of in vitro-fertilized (IVF) sheep embryos, the mRNA and protein expression levels of IL-6 and IL-6R were evaluated along with their localization, through real-time PCR, protein blotting, and immunohistochemistry. Specific patterns of IL-6 and IL-6R expression were recorded at both the mRNA and protein level at each stage of early ovine embryonic development. IL-6 was distributed primarily on the cytoplasm, however IL-6R was concentrated much more on the cell membrane. The role of IL-6 and IL-6R in the early ovine embryonic development was studied via the in vitro addition of recombinant human IL-6, at different concentrations and RNA interference. The addition of the recombinant IL-6 increased the embryonic IL-6 mRNA level, and the addition of 10ng/mL IL-6 significantly increased the embryo cleavage rate (p<0.05). IL-6 RNA interference effectively reduced the IL-6 protein level, but did not affect the rate of development of the IVF ovine embryos. This study provides a practical basis for further indepth studies on the mechanisms involved during early ovine embryonic development.

Anti-inflammatory activity of a water extract of Acorus calamus L. leaves on keratinocyte HaCaT cells

Ethnopharmacological relevance Acorus calamus L., sweet flag, is a well-known medicinal plant that grows worldwide wildly along swamps, rivers, and lakes. Aim of the study The aim of this study was to evaluate the anti-inflammatory activity of Acorus calamus leaf (ACL) extract and to explore its mechanism of action on human keratinocyte HaCaT cells. Materials and methods HaCaT cells treated with polyinosinic:polycytidylic acid (polyI:C) and peptidoglycan (PGN) induced the inflammatory reactions. The anti-inflammatory activities of ACL were investigated using RT-PCR, ELISA assay, immunoblotting, and immunofluorescence staining. Results HaCaT cells induced the pro-inflammatory cytokines, interleukin-8 (IL-8) and/or interleukin-6 (IL-6) expressions after treatment with polyI:C or PGN. ACL inhibited the expression of IL-8 and IL-6 RNA and protein levels, and attenuated the activation of NF-κB and IRF3 after polyI:C treatment. ACL also inhibited expression of IL-8 and activation of NF-κB following PGN induction. Conclusions These results suggest that ACL inhibits the production of pro-inflammatory cytokines through multiple mechanisms and may be a novel and effective anti-inflammatory agent for the treatment of skin diseases.

Interleukin-8 Expression Is Regulated by Histone Deacetylases through the Nuclear Factor-κB Pathway in Breast Cancer

We have reported recently that the chemokine interleukin 8 (IL-8)/CXCL8 was overexpressed in invasive estrogen receptor (ERalpha)-negative breast cancer cells compared with ERalpha-positive breast cancer cells. We now demonstrate that histone deacetylases (HDACs) play an essential role in the regulation of IL-8 gene expression in ERalpha-positive MCF-7 breast cancer cells. Treatment of MCF-7 cells with the HDAC inhibitor trichostatin A (TSA) led to a strong up-regulation of IL-8 protein and RNA levels in MCF-7 cells. The up-regulation of IL-8 in MCF-7 cells was time- and concentration-dependent. Moreover, run-on and transfection experiments demonstrated that IL-8 induction by HDAC inhibitors was transcriptional and involved mainly the nuclear factor-kappaB (NF-kappaB) site of the IL-8 promoter. These observations are corroborated by an up-regulation of NF-kappaB activity in MCF-7 cells in the presence of TSA. In addition, blocking NF-kappaB pathway by adenoviral delivery of a dominant-negative IkappaBorIkappaB kinase complex 2 (IKK2) mutant abolished IL-8 gene induction by histone deacetylase inhibitors. HDAC inhibitors triggered IKK phosphorylation and up-regulated p65 nuclear translocation, although they decreased the protein levels of IkappaBalpha, which accounts for NF-kappaB activation. TSA increased binding of acetylated histone 3 to the IL-8 gene promoter. In summary, our results demonstrate that NF-kappaB pathway repression by HDAC is responsible for the low expression of IL-8 in ERalpha-positive breast cancer cells.

Epstein-Barr Virus Lytic Transactivator Zta Enhances Chemotactic Activity through Induction of Interleukin-8 in Nasopharyngeal Carcinoma Cells

Epstein-Barr virus (EBV)-associated, undifferentiated type of nasopharyngeal carcinoma (NPC) is characterized by intensive leukocyte infiltration. Interaction between the infiltrating cells and the tumor cells has been considered crucial for NPC development. Recruitment of the infiltrates can be directed by certain chemokines present in the NPC tissues. It is unknown whether and how EBV lytic infection regulates expression of the chemokines. Using an antibody array, we first found that several chemokines secreted from EBV-infected NPC cells are increased upon EBV reactivation into the lytic cycle, and interleukin-8 (IL-8) is the chemokine upregulated most significantly and consistently. Further studies showed that the EBV lytic transactivator Zta is a potent inducer of IL-8 in NPC cells, augmenting secreted and intracellular IL-8 proteins, as well as IL-8 RNA. Zta upregulates Egr-1, a cellular transcription factor that has been involved in upregulation of IL-8, but the Zta-induced IL-8 expression is independent of Egr-1. The ability of Zta to transactivate the IL-8 promoter is important for the induction of IL-8, and we have identified two Zta-responsive elements in the promoter. Zta can bind to these two elements in vitro and can also be recruited to the IL-8 promoter in vivo. DNA-binding-defective Zta mutants can neither activate the IL-8 promoter nor induce IL-8 production. In addition, Zta-expressing NPC cells exert enhanced chemotactic activity that is mainly mediated by IL-8. Since IL-8 may contribute to not only leukocyte infiltration but also multiple oncogenic processes, the present study provides a potential link between EBV lytic infection and pathogenesis of NPC.

Essential role of RelA Ser311 phosphorylation by zetaPKC in NF-kappaB transcriptional activation.

The activation of the transcription factor NF-kappaB is central to the control of the cellular response triggered by many stimuli. Once released from the inhibitory molecule IkappaB, NF-kappaB is translocated to the nucleus, and it has to be phosphorylated to activate transcription. In zeta protein kinase C (PKC)-deficient cells, NF-kappaB is transcriptionally inactive and the phosphorylation of the RelA subunit in response to tumor necrosis factor (TNF-alpha) is severely impaired. In vitro assays showed that zetaPKC directly phosphorylates RelA. Here we demonstrate that Ser311 accounts for zetaPKC phosphorylation of RelA and that this site is phosphorylated in vivo in response to TNF-alpha. Also, an inactivating mutation of that residue severely impairs RelA transcriptional activity, blocks its anti-apoptotic function and abrogates the interaction of RelA with the co-activator CBP as well as its recruitment, and that of RNA polymerase II (Pol II) with the interleukin-6 (IL-6) promoter. The interaction of endogenous CBP with endogenous RelA is inhibited in zetaPKC-/- cells, as well as the binding of Pol II to the IL-6 promoter. These results demonstrate the mechanism whereby zetaPKC regulates NF-kappaB activation in vivo.

HIV type 1 infection alters cytokine mRNA expression in thymus.

We and others have previously shown that HIV infection of human thymus/liver implants in severe combined immunodeficient (SCID-hu Thy/Liv) mice results in a loss of CD4(+) thymocytes and a decreased recovery of human myeloid and erythroid colony-forming activity. Furthermore, our previous studies have shown that this decrease in colony-forming potential is due to indirect effects, rather than to massive loss of CD34(+) hematopoietic progenitor cells, suggesting that HIV infection might alter expression of hematopoietic cytokines. Herein we have investigated potential HIV-1-induced perturbations of hematolymphoid microenvironments by determining the effect of HIV-1 infection on levels of cytokine mRNAs in human stroma and thymocytes, using the reverse transcription-polymerase chain reaction (RT-PCR). The levels of interleukin 6 (IL-6), interferon gamma (IFN-gamma), and IL-2 RNAs increased and macrophage inflammatory protein 1beta (MIP-1beta) RNA decreased significantly in infected thymocytes. IL-6 RNA levels in stroma also increased somewhat with infection; however, expression of stromal cell-derived factor 1 (SDF-1) by stromal elements was not affected. IL-4 RNA levels were unaffected by infection in both stroma and thymocytes. Antiretroviral drug treatment of the infected animals, which results in restoration of colony-forming potential, tends to restore the cytokine mRNA levels in HIV-1-infected implants to those of mock-infected implants. These results indicate that HIV-1 infection can greatly distort the cytokine profiles in Thy/Liv implants, and suggest that cytokine perturbation could be involved in virus-induced inhibition of hematopoiesis.

Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjögren's syndrome keratoconjunctivitis sicca

PURPOSE. To compare epidermal growth factor (EGF) concentration in tear fluid and levels of inflammatory cytokines in the conjunctival epithelium of patients with Sjögren's syndrome keratoconjunctivitis sicca with those of normal controls. METHODS. Schirmer 1 tear testing, corneal fluorescein staining and conjunctival impression cytology for quantitation of goblet cell density were performed in ten patients with Sjögren's syndrome-associated keratoconjunctivitis sicca and ten asymptomatic normal controls. ELISA was used to detect the concentration of EGF in tear fluid and interleukin 6 in lysates of conjunctival cytology specimens obtained from all subjects. The levels of RNA transcripts encoding inflammatory cytokines [interleukin 1a_(IL-1a), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor a_(TNF-a), and transforming growth factor ß1 (TGF-ß1)] as well as a housekeeping gene (G3PDH) were evaluated in conjunctival cytology specimens taken from all subjects by semiquantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS. Decreased tear fluid EGF concentration was noted in Sjögren's syndrome patients (mean 0.68 ± 0.59 ng/ml) compared to controls (mean 1.66 ± 0.45 ng/ml, P = 0.004). Significantly increased levels of IL-1a, IL-6, IL-8, TNF-a and TGF-ß1 RNA transcripts were found in the conjunctival epithelium of Sjögren's syndrome patients compared to controls (P < 0.05), while the level of G3PDH was similar in both groups. The concentration of IL-6 protein was significantly higher in Sjögren's syndrome conjunctiva samples (P = 0.012). Tear EGF concentration correlated with Schirmer 1 scores (rho 0.767, P < 0.001), corneal fluorescein staining scores (rho -0.562, P = 0.01), conjunctival goblet cell density (rho 0.661, P = 0.001) and the levels of IL-1a_and IL-8 RNA in the conjunctival epithelium (rho -0.677 and -0.747, respectively, P = 0.001). Both IL-1a_and IL-8 RNA in the conjunctival epithelium increased as Schirmer 1 scores decreased (P = 0.001). IL-8 RNA level correlated with corneal fluorescein staining (rho 0.690, P = 0.001) and conjunctival goblet cell density (rho -0.767, P < 0.001). A significant decrease in IL-8 RNA level, corresponding to improvement in irritation symptoms and ocular surface disease, was observed in six eyes after two weeks of topical corticosteroid therapy. CONCLUSIONS. The balance of cytokines in the tear fluid and conjunctival epithelium is altered in Sjögren's syndrome. The severity of keratoconjunctivitis sicca in this condition increases as tear fluid EGF concentration decreases and levels of inflammatory cytokines in the conjunctival epithelium increase. These findings provide new insight into the pathogenesis of keratoconjunctivitis and provide potential targets for therapy.