Methylation of mammalian DNA by the DNA methyltransferase enzyme (dnmt-1) at CpG dinucleotide sequences has been recognized as an important epigenetic control mechanism in regulating the expression of cellular genes (Yen, R. W., Vertino, P. M., Nelkin, B. D., Yu, J. J., el-Deiry, W., Cumaraswamy, A., Lennon, G. G., Trask, B. J., Celano, P., and Baylin, S. B. (1992) Nucleic Acids Res. <em>20</em>, 2287-2291; Ramchandani, S., Bigey, P., and Szyf, M. (1998) Biol. Chem. 379, 535-5401). Here we show that interleukin (<em>IL</em>)-6 regulates the methyltransferase promoter and resulting enzyme activity, which requires transcriptional activation by the Fli-1 transcription factor (Spyropoulos, D. D., Pharr, P. N., Lavenburg, K. R., Jackers, P., Papas, T. S., Ogawa, M., and Watson, D. K. (1998) Mol. Cell. Biol. 15, 5643-5652). The data suggest that inflammatory cytokines such as <em>IL</em>-6 may exert many epigenetic changes in cells via the regulation of the methyltransferase gene. Furthermore, <em>IL</em>-6 regulation of transcription factors like Fli-1, which can help to direct cells along opposing differentiation pathways, may in fact be reflected in part by their ability to regulate the methylation of cellular genes.

Human <em>IL</em>-10 (h<em>IL</em>-10) modulates critical immune and inflammatory responses by way of interactions with its high- (<em>IL</em>-10R1) and low-affinity (<em>IL</em>-10R2) cell surface receptors. Human cytomegalovirus exploits the <em>IL</em>-10 signaling pathway by expressing a functional viral <em>IL</em>-10 homolog (cmv<em>IL</em>-10), which shares only 27% sequence identity with h<em>IL</em>-10 yet signals through <em>IL</em>-10R1 and <em>IL</em>-10R2. To define the molecular basis of this virus-host interaction, we determined the 2.7-A crystal structure of cmv<em>IL</em>-10 bound to the extracellular fragment of <em>IL</em>-10R1 (s<em>IL</em>-10R1). The structure reveals cmv<em>IL</em>-10 forms a disulfide-linked homodimer that binds two s<em>IL</em>-10R1 molecules. Although cmv<em>IL</em>-10 and h<em>IL</em>-10 share similar intertwined topologies and s<em>IL</em>-10R1 binding sites, their respective interdomain angles differ by approximately 40 degrees. This difference results in a striking re-organization of the <em>IL</em>-10R1s in the putative cell surface complex. Solution binding studies show cmv<em>IL</em>-10 and h<em>IL</em>-10 share essentially identical affinities for s<em>IL</em>-10R1 whereas the Epstein-Barr virus <em>IL</em>-10 homolog (ebv<em>IL</em>-10), whose structure is highly similar to h<em>IL</em>-10, exhibits a approximately <em>20</em>-fold reduction in s<em>IL</em>-10R1 affinity. Our results suggest cmv<em>IL</em>-10 and ebv<em>IL</em>-10 have evolved different molecular mechanisms to engage the <em>IL</em>-10 receptors that ultimately enhance the respective ability of their virus to escape immune detection.

Interleukin-1 (<em>IL</em>-1) has been shown to exert profibrotic activity in a number of disease models, including crescentic glomerulonephritis and pulmonary fibrosis, but the mechanisms by which this operates are poorly understood. Recent studies have identified a novel mechanism promoting renal fibrosis: tubular epithelial-myofibroblast transdifferentiation (TEMT). The present study examined whether <em>IL</em>-1 can stimulate TEMT in vitro. Cells of the normal rat kidney tubular epithelial cell line (NRK52E) were grown to confluence on collagen-coated plates and cultured for 5 days in the presence 1 to <em>20</em> ng/mL of <em>IL</em>-1alpha. Doses of 10 to <em>20</em> ng/mL of <em>IL</em>-1 caused transdifferentiation of NRK52E cells into myofibroblast-like cells. Scanning electron microscopy identified <em>IL</em>-1-induced morphological changes as a loss of apical-basal polarity and microvilli, cell hypertrophy, and the development of an elongated and invasive appearance. Phenotypically, <em>IL</em>-1-induced TEMT was characterized by de novo messenger RNA and protein expression of the mesenchymal marker alpha-smooth muscle actin, shown by Northern blotting, immunohistochemistry, and Western blotting. This was accompanied by loss of the epithelial marker E-cadherin. The addition of an excess of <em>IL</em>-1-receptor antagonist completely inhibited <em>IL</em>-1-induced TEMT. <em>IL</em>-1 was shown to stimulate the secretion of active transforming growth factor-beta1 (TGF-beta1) by NRK52E cells. Furthermore, the addition of a neutralizing anti-TGF-beta1 antibody inhibited <em>IL</em>-1-induced TEMT. In conclusion, <em>IL</em>-1 is a profibrogenic cytokine capable of inducing TEMT through a TGF-beta1-dependent mechanism. This may represent a novel mechanism by which <em>IL</em>-1 induces renal fibrosis in vivo.

<em>IL</em>-17A is a cytokine secreted by the newly described Th17 cells implicated in rheumatoid arthritis (RA). Less is known about its receptors in synoviocytes. <em>IL</em>-17RA and <em>IL</em>-17RC were found to be overexpressed in RA peripheral whole blood and their expression was detected locally in RA synovium. In vitro, <em>IL</em>-17A synergized with TNF-alpha to induce <em>IL</em>-6, <em>IL</em>-8, CCL-<em>20</em>, and matrix metalloproteinase-3. Using microarrays, a specific up-regulation of Glu-Leu-Arg+ CXC chemokines was observed in <em>IL</em>-17A-treated synoviocytes. Using both posttranslational inhibitions by silencing interfering RNA and extracellular blockade by specific inhibitors, we showed that both <em>IL</em>-17RA and <em>IL</em>-17RC are implicated in <em>IL</em>-17A-induced <em>IL</em>-6 secretion, whereas in the presence of TNF-alpha, the inhibition of both receptors was needed to down-regulate <em>IL</em>-17A-induced <em>IL</em>-6 and CCL-<em>20</em> secretion. Thus, <em>IL</em>-17A-induced <em>IL</em>-6, <em>IL</em>-8, and CCL<em>20</em> secretion was dependent on both <em>IL</em>-17RA and <em>IL</em>-17RC, which are overexpressed in RA patients. <em>IL</em>-17A-induced pathogenic effects may be modulated by <em>IL</em>-17RA and/or <em>IL</em>-17RC antagonism.

The activity of the steroid-inducible protein lipocortin-1 (LC1; with a primary sequence of 346 amino acids; also called annexin 1), a fragment corresponding to amino acids 1-188 and a short peptide from the N-terminus (amino acid 2-26) were tested for anti-inflammatory actions in three models of acute inflammation in the mouse in comparison with a mAb anti-CD11b (alpha CD11b). In the mouse air-pouch model LC1, fragment 1-188 and peptide Ac2-26 exhibited powerful inhibitory effects (ED50 approximately 5.2, 38 and 88 micrograms/mouse, respectively) on leukocyte migration elicited by <em>IL</em>-1. LC1 was approximately <em>20</em>0 times more potent than Ac2-26 on a molar basis although both gave maximal inhibitions, in contrast fragment 1-188 only produced a partial dose-response curve. LC1 was approximately <em>20</em> times more potent on a molar basis in this assay than the alpha CD11b mAb. Peptide Ac2-26 and the mAb alpha CD11b also blocked cell migration into the air-pouch induced by <em>IL</em>-8 with approximately the same potency. In the mouse skin edema and zymosan peritonitis assays Ac2-26 was inhibitory (ED50 of <em>20</em>0 micrograms/mouse) but less so than the alpha CD11b antibody (ED50 approximately 0.5 mg/mouse). Both LC1 (10 micrograms) and Ac2-26 (<em>20</em>0 micrograms) completely blocked FMLP-induced neutropenia in the mouse. Studies using an inactivated LC1 preparation, which binds to the same high affinity binding sites as the biologically active material, indicated that the short peptide acts on the same sites as the native LC1. This study confirms the activity of LC1 in another model of experimental inflammation and suggests that it acts partly through inhibition of leukocyte activation with an overall effect qualitatively comparable to the blocking of CD11b portion of a beta 2-integrin complex. It also shows that peptides derived from the N-terminal domain of LC1 may mimic the activity of the full length molecule and points the way for a new family of anti-inflammatory substances that inhibit leukocyte trafficking.

Acute liver failure (ALF) shares striking similarities with septic shock where a decrease in HLA-DR expression on monocytes is associated with disease severity and predicts outcome. We investigated monocyte HLA-DR expression in ALF in relation to inflammatory mediator levels and clinical outcome. Monocyte HLA-DR expression was determined in 50 patients with acetaminophen-induced ALF (AALF) and <em>20</em> non-acetaminophen-induced ALF (NAALF). AALF patients were divided into dead/transplanted (AALF-NS, n = 26) and spontaneous survivors (AALF-S, n = 24). Fifty patients with chronic liver disease (CLD) and 50 healthy volunteers served as controls. Monocyte HLA-DR expression was determined by double-color flow-cytometry with monoclonal antibodies detecting HLA-DR and monocyte specific CD14. Serum levels of interleukin (<em>IL</em>) -4, -6, -10, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were concomitantly measured by ELISA. Compared to healthy volunteers (75%) and CLD (67%) monocyte HLA-DR percentage expression was lower in AALF (15%, P < .001) and NAALF (22 %, P < .001). Compared to AALF-S, AALF-NS had lower monocyte HLA-DR % (11% vs. 36%, P < .001) and higher levels of <em>IL</em>-4, <em>IL</em>-6, <em>IL</em>-10 and TNF-alpha (P < .001). HLA-DR percentage negatively correlated with INR, blood lactate, pH and levels of encephalopathy (r = -0.8 to -0.5, P < .01), <em>IL</em>-10 (r = -0.8, P < .0001), TNF-alpha (r = -0.4, P = .02). HLA-DR percentage level <or=15% has a 96% sensitivity and 100% specificity and 98% accuracy in predicting poor prognosis. In conclusion, the strong relationship of monocyte HLA-DR expression with indices of disease severity, mediators of inflammation and outcome indicates a key role for this molecule as a biomarker of disease severity and prognosis.

High linoleic acid (LA) intakes have been suggested to reduce alpha-linolenic acid [ALA, 18:3(n-3)] metabolism to eicosapentaenoic acid [EPA, <em>20</em>:5(n-3)] and docosahexaenoic acid [DHA, 22:6(n-3)], and favor high arachidonic acid [ARA, <em>20</em>:4(n-6)]. We used a randomized cross-over study with men (n = 22) to compare the effect of replacing vegetable oils high in LA with oils low in LA in foods, while maintaining constant ALA, for 4 wk each, on plasma (n-3) fatty acids. Nonvegetable sources of fat, except fish and seafoods, were unrestricted. We determined plasma phospholipid fatty acids at wk 0, 2, 4, 6, and 8, and triglycerides, cholesterol, serum CRP, and <em>IL</em>-6, and platelet aggregation at wk 0, 4, and 8. LA and ALA intakes were 3.8 +/- 0.12% and 1.0 +/- 0.05%, and 10.5 +/- 0.53% and 1.1 +/- 0.06% energy with LA:ALA ratios of 4:0 and 10:1 during the low and high LA diets, respectively. The plasma phospholipid LA was higher and EPA was lower during the high than during the low LA diet period (P < 0.001), but DHA declined over the 8-wk period (r = -0.425, P < 0.001). The plasma phospholipid ARA:EPA ratios were (mean +/- SEM) <em>20</em>.7 +/- 1.52 and 12.9 +/- 1.01 after 4 wk consuming the high or low LA diets, respectively (P < 0.001); LA was inversely associated with EPA (r = -0.729, P < 0.001) but positively associated with ARA:EPA (r = 0.432, P < 0.001). LA intake did not influence ALA, ARA, DPA, DHA, or total, LDL or HDL cholesterol, CRP or <em>IL</em>-6, or platelet aggregation. In conclusion, high LA intakes decrease plasma phospholipid EPA and increase the ARA:EPA ratio, but do not favor higher ARA.

Aberrant expression of FLT3 has been found in most cases of B-lineage ALL and AML, and subsets of T cell ALL, CML in blast crisis and CLL. In <em>20</em>% of patients with AML the receptor has small internal tandem duplications of the juxtamembrane region which appear to contitutively activate the receptor. To investigate whether FLT3 activation could play a role in leukemia, we generated a constitutively activated FLT3 by fusing its cytoplasmic domain to the helix-loop-helix domain of TEL in analogy to the fusion that occurs with TEL-PDGFR in CMML. In vitro translation assays demonstrated oligomerization and intrinsic tyrosine kinase activity of the TEL-FLT3 chimeric receptor. Constitutively activated TEL-FLT3 conferred <em>IL</em>-3 independence and long-term proliferation to transfected Ba/F3 cells. Immunoblot analyses showed that JAK 2, STAT 3, STAT 5a, STAT 5b and CBL were tyrosine-phosphorylated in TEL-FLT3 expressing Ba/F3 cells in the absence of <em>IL</em>-3. These data suggest a possible role for the JAK/STAT pathway in FLT3 signaling. Transplantation of TEL-FLT3 expressing Ba/F3 cells into syngeneic mice caused mortality in all mice by 3 weeks after injection. Histopathologic analysis demonstrated a massive infiltration of mononuclear cells in the liver, spleen and bone marrow. The mimicking of naturally occurring TEL fusions provides an approach to assess aspects of the biology of activated FLT3, or other receptor-type tyrosine kinases (RTKs) in leukemic transformation.

It has been recently demonstrated that conditioned media from Con A-stimulated splenocyte cultures contain a novel lymphokine termed <em>IL</em> 3. <em>IL</em> 3 is characterized by its ability to induce <em>20</em>-alpha-hydroxysteroid dehydrogenase in spleen cells from young nu/nu mice. In search of a convenient source for biochemical and in vivo studies of this lymphokine, a number of established murine cell lines were screened for the constitutive and induced production of <em>Il</em> 3. It was found that WEHI-3 cells, originally designated as a myelomonocyte cell line, produced high levels of <em>IL</em> 3 constitutively. Added mitogen and/or phorbol acetate did not enhance the production of <em>IL</em> 3. Production of <em>IL</em> 3 varied among various sublines of WEHI-3. <em>IL</em> 3 purified from WEHI-3-conditioned media has biochemical and biologic characteristics identical to those obtained from Con A-conditioned media. WEHI-3 conditioned media, however, generally contained 100-fold higher levels of <em>IL</em> 3 than conditioned media from Con A-stimulated splenic lymphocytes.

The in vivo production of interleukin (<em>IL</em>)-10, <em>IL</em>-6, <em>IL</em>-2, and tumor necrosis factor (TNF)-alpha in tumor samples was investigated by immunohistochemistry in 54 non-Hodgkin's lymphomas (NHLs). Respectively, 55, 89, 23, and 29% of tumor samples were found positive for <em>IL</em>-10, <em>IL</em>-6, <em>IL</em>-2, and TNF-alpha expression by immunohistochemistry. Using reverse transcription-PCR, the mRNA of <em>IL</em>-10 and <em>IL</em>-6 were detectable in all samples tested and in 90 and 34% of the samples for TNF-alpha and <em>IL</em>-2, respectively. In 13 patients, fresh tumor tissue was available for B NHL cell purification with Dynabeads. <em>IL</em>-10, <em>IL</em>-6, <em>IL</em>-2, and TNF-alpha were detectable in the supernatant of 38, 100, 0, and 23% of purified tumor cell preparations (PTCPs), respectively. All patients with detectable <em>IL</em>-10 in culture had increased serum <em>IL</em>-10. <em>IL</em>-6 production by tumor cells and serum <em>IL</em>-6 levels were also found to be highly correlated (P < 0.0001). This suggests that tumor cells are a major source of serum <em>IL</em>-1O and <em>IL</em>-6 in these patients. Exogenous <em>IL</em>-10, <em>IL</em>-6, <em>IL</em>-2, and TNF-alpha significantly enhanced the [3H]thymidine uptake in 13 of 13 (100%), 5 of 13 (38%), 9 of 13 (69%), and 2 of 10 (<em>20</em>%) PTCPs costimulated with anti-CD40, respectively. <em>IL</em>-2, <em>IL</em>-6, and TNF-alpha synergized with <em>IL</em>-10 in 54, 23, and 30% of PTCPs. The combination of <em>IL</em>-10, <em>IL</em>-2, and <em>IL</em>-6 induced the maximal level of proliferation in 12 (92%) of 13 PTCPs. CD40 ligand mRNA expression was also detectable in vivo using reverse transcription-PCR in 28 of the 29 (97%) tumor samples tested, including 11 of those tested for [3H]thymidine incorporation. These results show that <em>IL</em>-1O, <em>IL</em>-6, <em>IL</em>-2, and TNF-alpha are produced in NHL tumors and may cooperate in vivo to increase NHL cell proliferation.

The family of <em>IL</em>-10-related cytokines includes several human members, <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, <em>IL</em>-24 and <em>IL</em>-26, and a series of herpesviral and poxviral paralogs. Some of these cytokines share common receptor subunits. In this study, we investigated the effects of these cytokines on naive T cell differentiation, antigen-specific T cell suppression, survival ad expression of surface markers in comparison to <em>IL</em>-10 and cytomegalovirus (CMV)-<em>IL</em>-10. Human CD45RA(+) T cells were stimulated in the presence of <em>IL</em>-10-family cytokines in sequential 12-day cycles. After three to four cycles of stimulation, <em>IL</em>-10 and CMV-<em>IL</em>-10 led to increased IFN-gamma and <em>IL</em>-10 but decreased <em>IL</em>-4 and <em>IL</em>-13. Interestingly, long-term exposure of T cells to <em>IL</em>-19, <em>IL</em>-<em>20</em> and <em>IL</em>-22 down-regulated IFN-gamma but up-regulated <em>IL</em>-4 and <em>IL</em>-13 in T cells and supported the polarization of naive T cells to Th2-like cells. In contrast, neutralization of endogenous <em>IL</em>-22 activity by <em>IL</em>-22-binding protein decreased <em>IL</em>-4, <em>IL</em>-13 and IFN-gamma synthesis. The antigen-specific suppressor activity of <em>IL</em>-10 and CMV-<em>IL</em>-10 was not observed for any of the other <em>IL</em>-10-family cytokines. These data demonstrate that <em>IL</em>-19, <em>IL</em>-<em>20</em> and <em>IL</em>-22 may participate in T cell-mediated diseases by distinct regulation of T cell cytokine profiles.

Since unmethylated CpG motifs are more frequent in DNA from bacteria than vertebrates, and the unmethylated CpG motif has recently been reported to have stimulatory effects on lymphocytes, we speculated that bacterial DNA may induce inflammation in the lower respiratory tract through its content of unmethylated CpG motifs. To determine the role of bacterial DNA in lower airway inflammation, we intratracheally instilled prokaryotic and eukaryotic DNA in C3H/HeBFEJ mice and performed whole lung lavage 4 h after the exposure. Heat denatured, single stranded Escherichia coli genomic DNA (0.06 ng endotoxin/microg DNA) was compared to heat denatured, single stranded calf thymus DNA (0.007 endotoxin/microg DNA). 10 microg of bacterial DNA, in comparison to 10 microg of calf thymus DNA, resulted in a fourfold increase in the concentration of cells (P = 0.0002), a fivefold increase in the concentration of neutrophils (P = 0.0002), a 50-fold increase in the concentration of TNF-alpha (P = 0.001), and a fourfold increase in the concentration of both <em>IL</em>-6 (P = 0.0003) and macrophage inflammatory protein-2 (P = 0.0001) in the lavage fluid. Importantly, instillation of 0.60 ng of E. coli LPS resulted in a negligible inflammatory response. To test whether the stimulatory effects of bacterial DNA are due to its unmethylated CpG dinucleotides, we methylated the bacterial DNA and also prepared <em>20</em> base pair oligonucleotides with and without CpG motifs. In comparison to instillation of untreated bacterial DNA, methylation of the bacterial DNA resulted in a significant reduction in the concentration of cells and cytokines in the lower respiratory tract. Moreover, oligonucleotides containing embedded unmethylated CpG motifs resulted in inflammation in the lower respiratory tract that was indistinguishable from that observed with untreated bacterial DNA. In contrast, oligonucleotides without the embedded CpG motifs or with embedded but methylated CpG motifs resulted in significantly less inflammation in the lower respiratory tract. The possible relevance of these data to human disease was shown by extracting and analyzing DNA in sputum from patients with cystic fibrosis (CF). Approximately 0.1 to 1% of this sputum DNA was bacterial. Intratracheal instillation of highly purified CF sputum DNA caused acute inflammation similar to that induced by bacterial DNA. These findings suggest that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic role in inflammatory lung disease.

OBJECTIVE

Anti-tumor necrosis factor a (anti-TNFalpha) therapy has shown efficacy in the treatment of rheumatoid arthritis (RA). Since interleukin-1 (IL-1), TNFalpha, and IL-17 have many additive and/or synergistic effects in vitro, we tested whether their combined inhibition by soluble receptors would lead to an enhanced effect on ex vivo models of synovial inflammation and bone destruction.

METHODS

RA synovium and bone explants were cultured for 7 days in the presence of 1 microg/ml soluble TNFalpha receptor (STNFR; as in current therapy), type II soluble IL-1 receptor (sIL-1RII), or sIL-17R either alone or in combination. Their effects on the production of IL-6 and the release of C-telopeptide of type I collagen (CTX), a marker of type I collagen destruction, were measured by enzyme-linked immunosorbent assay.

RESULTS

In synovium, each soluble receptor alone decreased IL-6 production and CTX release by approximately 35% and approximately 55%, respectively. The combination of all 3 receptors was more effective, inhibiting IL-6 production and collagen degradation by up to 70%. Neither sIL-17R, sIL-1RII, or sTNFR alone had no effect (or an effect of <20% inhibition) on IL-6 production in 18%, 33%, and 22%, respectively, of the samples. In bone, sIL-17R, sIL-1RII, and sTNFR decreased IL-6 production by 23%, 50%, and 37%, respectively, while the combination decreased IL-6 production by 75%. A 50% inhibition of CTX release was obtained with sIL-1RII for 63% of the samples versus 38% of the samples with either sTNFR or sIL-17R. However, the combination of all 3 receptors was not more potent than sIL-1RII alone.

CONCLUSIONS

The inhibitory effect of sTNFR on IL-6 production and collagen degradation in RA synovium and bone was increased in combination with sIL-17R and sIL-1RII. These results support the concept of combination therapy, which may increase the percentage of responding patients as well as the degree of individual patient response.

BACKGROUND

Obesity increases the risk for iron deficiency, but the underlying mechanism is unclear. It is possible that overweight individuals may have lower dietary iron intake and/or bioavailability. Alternatively, obesity-related inflammation may increase hepcidin concentrations and reduce iron availability. Circulating hepcidin levels have not been compared in normal weight vs overweight individuals.

OBJECTIVE

The objective of this study was to compare iron status, dietary iron intake and bioavailability, as well as circulating levels of hepcidin, leptin and interleukin-6 (IL-6), in overweight vs normal weight children.

METHODS

In 6-14-year-old normal and overweight children (n=121), we measured dietary iron intake, estimated iron bioavailability and determined body mass index s.d. scores (BMI-SDS). In all children (n=121), we measured fasting serum ferritin, soluble transferrin receptor (sTfR), C-reactive protein (CRP) and leptin; in a subsample, we measured IL-6 (n=68) and serum hepcidin (n=30).

RESULTS

There were no significant differences in dietary iron intake or bioavailability comparing normal and overweight children. The prevalence of iron-deficient erythropoiesis (an increased sTfR concentration) was significantly higher in the overweight than in the normal weight children (20 vs 6%, P=0.022, with sTfR concentrations of 4.40+/-0.77 and 3.94+/-0.88 mg l(-1), respectively, P=0.010). Serum hepcidin levels were significantly higher in the overweight children (P=0.001). BMI-SDS significantly correlated with sTfR (P=0.009), serum hepcidin (P=0.005) and the three measures of subclinical inflammation, namely CRP (P<0.001), IL-6 (P<0.001) and leptin (P<0.001). In a multiple regression model, serum hepcidin was correlated with BMI-SDS (P=0.020) and body iron (P=0.029), but not with the inflammatory markers.

CONCLUSIONS

Our findings indicate that there is reduced iron availability for erythropoiesis in overweight children and that this is unlikely due to low dietary iron supply but rather due to hepcidin-mediated reduced iron absorption and/or increased iron sequestration.

Molecular and cellular understanding of psoriasis pathogenesis has evolved considerably over the last 30 years beginning in the early 1980s when psoriasis was thought to be a skin disease driven by keratinocyte hyperproliferation. During the next <em>20</em> years, the role of the immune system and T-helper (Th) cells in psoriasis pathogenesis was recognized. The presence of the interleukin (<em>IL</em>)-12 cytokine in psoriatic lesions led to the postulate that psoriasis is mediated by Th1 cells. Recent evidence has revealed a role for Th17 cells, and other immune cells, as proximal regulators of psoriatic skin inflammation. <em>IL</em>-17A, the principal effector cytokine of Th17 cells, stimulates keratinocytes to produce chemokines, cytokines, and other proinflammatory mediators thereby enabling <em>IL</em>-17A to bridge the innate and adaptive immune systems to sustain chronic inflammation. This model underlies the rationale for inhibiting <em>IL</em>-17A signaling as a potential therapeutic approach to disrupt the psoriatic inflammatory loop. Several monoclonal antibodies that inhibit the <em>IL</em>-17 pathway are in clinical development. These agents exhibit promising clinical efficacy and tolerability profiles including immunohistochemical improvement in psoriatic plaques. Results from clinical trials with <em>IL</em>-17 pathway inhibitors are refining our understanding of psoriasis pathogenesis and may provide a new therapeutic approach for patients with moderate to severe psoriasis.

Mycobacterium tuberculosis and its components have been shown to stimulate mononuclear phagocytes in vitro to release interleukin-1 beta (<em>IL</em>-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (<em>IL</em>-6). Animal models of tuberculosis (TB) also demonstrate the presence of cytokines in granulomas. We hypothesized that bronchoalveolar lavage (BAL) cells from patients with pulmonary TB would have increased spontaneous release of <em>IL</em>-1 beta, <em>IL</em>-6, and TNF-alpha and would have a concomitant alveolitis. We performed BAL on 26 patients with active TB and on six normal volunteers. BAL fluid from radiographically involved and uninvolved sites was evaluated separately for cell types and the spontaneous release of cytokines. The alveolar inflammation in involved sites was characterized by an increase in lymphocytes (miliary TB, 38 +/- 10%; involved sites, 22 +/- 4%; uninvolved sites, 13 +/- 2%; normal, 5 +/- 2%) and neutrophils (involved sites, 21 +/- 7%; uninvolved sites, 3 +/- 2%). There was a significant increase in the spontaneous release of <em>IL</em>-1 beta (501 +/- 280 pg/ml), TNF-alpha (782 +/- 165 pg/ml), and <em>IL</em>-6 (473 +/- 157 pg/ml) from involved sites of TB patients that was 5- to <em>20</em>-fold greater than uninvolved sites, normal controls, or miliary TB. Northern analysis revealed increased gene expression of <em>IL</em>-1 beta, TNF-alpha, and <em>IL</em>-6 from the involved sites from two patients with TB compared with two negative controls. We conclude that BAL cells, especially alveolar macrophages, are activated in the alveolar inflammation of active TB and spontaneously release increased quantities of <em>IL</em>-1 beta, <em>IL</em>-6, and TNF-alpha, and that these cytokines are likely to be involved in directing granuloma formation and control of M. tuberculosis infection.

Mononuclear cell infiltration and local cytokine elaboration are hallmarks of inflammatory and immunologic heart diseases. To test the hypothesis that cytokines can modulate cardiac myocyte growth and phenotype, myocytes cultured from neonatal rat hearts were exposed to <em>IL</em>-1 beta, an inflammatory cytokine prevalent in myocardial inflammation. <em>IL</em>-1 beta (2 ng/ml, 24 h) increased [3H]leucine incorporation by 30 +/- 4% (P < 0.001, n = 29) and net cellular protein content by <em>20</em> +/- 4% (P < 0.001, n = 27), but had no effect on DNA synthesis. Northern hybridization showed that <em>IL</em>-1 beta increased prepro-atrial natriuretic factor (ANF) mRNA (5.8 +/- 1.5-fold, P < 0.01, n = 13) and beta-myosin heavy chain (beta-MHC) mRNA >> 10-fold, n = 4), and decreased mRNA levels for sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) (-46 +/- 7%; P < 0.001; n = 11), calcium release channel (CRC) (-65 +/- 11%, P < 0.001, n = 8) and voltage-dependent calcium channel (VDCC) (-53 +/- 7%, P < 0.001, n = 8). NG-monomethyl-L-arginine (1 mM), an inhibitor of nitric oxide (NO) synthesis, did not inhibit the <em>IL</em>-1 beta-induced protein synthesis or changes in mRNA levels. In ventricular myocardium obtained from adult rats treated with lipopolysaccharide (4 mg/kg intraperitoneally 18 h) to stimulate systemic cytokine production, there were changes in the mRNA levels for beta-MHC (6 +/- 1-fold, P < 0.01, n = 4), SERCA2 (-65 +/- 4%, P < 0.0001, n = 4), CRC (-67 +/- 5%, P < 0.001, n = 4), and VDCC (-58 +/- 5%, P < 0.001; n = 4) that were qualitatively similar to those observed in cultured myocytes. Thus, <em>IL</em>-1 beta, acting via an NO-independent mechanism, caused myocyte hypertrophy associated with induction of fetal genes (ANF and beta-MHC) and downregulation of three important calcium regulatory genes (SERCA2, CRC, and VDCC). <em>IL</em>-1 beta may contribute to the abnormal structural and functional alterations of cardiac myocytes in conditions marked by mononuclear cell infiltration.

OBJECTIVE

We report two clinical trials in non-small cell lung cancer (NSCLC) patients evaluating immune response, toxicity, and clinical outcome after vaccination with the telomerase peptide GV1001: a phase II trial (CTN-<em>20</em>06) in patients vaccinated after chemoradiotherapy and an 8-year update on a previously reported phase I/II trial (CTN-<em>20</em>00).

METHODS

CTN-<em>20</em>06: 23 inoperable stage III patients received radiotherapy (2 Gy × 30) and weekly docetaxel (<em>20</em> mg/m(2)), followed by GV1001 vaccination. CTN-<em>20</em>00: 26 patients were vaccinated with two telomerase peptides (GV1001 and I540). The immune responses were evaluated by T-cell proliferation and cytokine assays.

RESULTS

CTN-<em>20</em>06 trial: a GV1001-specific immune response developed in 16/<em>20</em> evaluable patients. Long-term immunomonitoring showed persisting responses in 13 subjects. Serious adverse events were not observed. Immune responders recorded a median PFS of 371 days, compared with 182 days for nonresponders (P = 0.<em>20</em>). CTN-<em>20</em>00 trial update: 13/24 evaluable subjects developed a GV1001 response. The immune responders achieved increased survival compared with nonresponders (median 19 months vs. 3.5 months; P < 0.001). Follow-up of four long-time survivors showed that they all harbored durable GV1001-specific T-cell memory responses and IFNγ(high)/IL-10(low)/IL-4(low) cytokine profiles. Two patients are free of disease after 108 and 93 months, respectively.

CONCLUSIONS

Vaccination with GV1001 is well tolerated, immunizes the majority of NSCLC patients and establishes durable T-cell memory. The considerable immune response rate and low toxicity in the phase II trial support the concept of combining chemoradiotherapy with vaccination. The survival advantage observed for immune responders warrants a randomized trial.

BACKGROUND

Association between inflammatory markers and intermuscular adipose tissue (IMAT) has been reported. We hypothesized that subclinical inflammation of adipose tissue surrounding and infiltrating muscle could be related to the metabolic and functional abnormalities of the "aging muscle."

METHODS

In <em>20</em> healthy elderly men undergoing elective vertebral surgery, IMAT within erector spinae was evaluated by magnetic resonance imaging and body composition by dual-energy x-ray absorptiometry. Fasting glucose, insulin, high-sensitive C-reactive protein (hs-CRP), leptin, adiponectin, and interleukin 6 (<em>IL</em>-6) were measured, and insulin resistance was estimated by homeostasis model assessment (HOMA) index. In subcutaneous adipose tissue (SAT) biopsies near the erector spinae, quantification of gene expression was performed.

RESULTS

IMAT showed a significant association with body mass index and total and regional body fat, even after adjustment for age. Insulin, HOMA, and leptin were significantly correlated with IMAT, whereas hs-CRP presented an association of borderline significance. IL-6 expression in SAT was significantly associated with IMAT; IL-6 messenger RNA (mRNA) was negatively associated with adiponectin and peroxisome proliferator-activated receptor gamma expression. In multivariate regression analysis, 68% of IMAT variance was explained by fat mass and age, independent of waist circumference, leptin, HOMA, and IL-6 mRNA.

CONCLUSIONS

IMAT was primarily related to age and total body adiposity; subclinical inflammation in fat significantly contributes to IMAT.

We investigated the effect of OKT3 antibody and interleukin 2 (<em>IL</em>-2) on Tac antigen expression and the proliferation of human peripheral blood mononuclear leukocytes. OKT3 monoclonal antibody at low, nonmitogenic concentrations (25 pg/ml) or <em>IL</em>-2 alone at optimal concentrations (<em>20</em> U/ml) did not induce <em>IL</em>-2 receptor expression, as measured by Tac antibody or by T cell proliferation. However, costimulation with these concentrations of OKT3 antibody and <em>IL</em>-2 led to Tac antigen expression and T cell proliferation. These data suggest that the T cells are activated in two steps: OKT3 antibody at 25 pg/ml does not induce Tac antigen expression, but preactivates T cells to become responsive to <em>IL</em>-2. The addition of exogenous <em>IL</em>-2 then leads to expression of the <em>IL</em>-2 receptor, as recognized by Tac antibody, and to subsequent proliferation.

Interleukin-2 (<em>IL</em>-2) is a T-cell-derived polypeptide hormone of 133 amino acids which exerts its growth-promoting activity via a surface receptor. Originally, <em>IL</em>-2 was believed to be a unique growth factor for activated T cells; more recent studies, however, have demonstrated that certain B-cell tumours as well as normal activated B lymphocytes express a surface molecule which is recognized by monoclonal antibodies directed against the <em>IL</em>-2 receptor. Furthermore, we and others have shown recently that activated B cells proliferate in response to either immunoaffinity-purified or recombinant <em>IL</em>-2. These controversial findings prompted us to undertake a detailed quantitative comparison of <em>IL</em>-2 receptor expression on activated B and T cells. We show here, using biosynthetically labelled <em>IL</em>-2(3H-<em>IL</em>-2) and anti-<em>IL</em>-2 receptor antibody (3H-PC61) that activated B and T cells express both high-affinity (apparent dissociation constant, Kd approximately <em>20</em> pM) and low-affinity (Kd approximately 1,000 pM) <em>IL</em>-2 receptors. Binding of <em>IL</em>-2 to both classes of receptor is inhibited by the monoclonal anti-<em>IL</em>-2 receptor antibody PC61. B blasts express half as many total <em>IL</em>-2 binding sites or PC61 binding sites as T blasts, and the ratio of the number of low- to high-affinity receptors for each cell type is approximately 10:1. Immunoprecipitation analysis of surface-labelled blasts indicates that B and T cells have <em>IL</em>-2 receptors of similar relative molecular mass. Taken together, these data suggest strongly that <em>IL</em>-2 can act as a growth hormone for both B and T lymphocytes.

This study determined the presence of interleukin 1 (<em>IL</em>-1), interleukin 6 (<em>IL</em>-6), tumour necrosis factor alpha (TNF alpha), tumour necrosis factor beta (TNF beta), interferon gamma (IFN gamma), transforming growth factor beta 2 (TGF beta 2) and fibroblast proliferation activity (FPA) in vitreous aspirates from eyes undergoing vitrectomy for the treatment of retinal detachment complicated by proliferative vitreoretinopathy (PVR) or uncomplicated retinal detachment (RD). Cadaveric vitreous from normal subjects were used as controls. The results showed that <em>IL</em>-1 and <em>IL</em>-6 predominated in vitreous from eyes with PVR or RD, and that concentrations of <em>IL</em>-6 greater than <em>20</em> pg/ml were more frequently found in PVR than in RD (p = 0.031) or control specimens (p = 0.006). Low levels of TNF alpha were observed in 4/18 eyes with PVR, 1/15 eyes with RD and 1/15 control vitreous, and small concentrations of TNF alpha were seen in 3/18 eyes with PVR, 1/15 eyes with RD and 2/15 control vitreous. IFN gamma was detected in 12/18 eyes with PVR, but only in 5/15 eyes with RD (p = 0.048) and 6/15 control specimens. TGF beta 2 was present in all vitreous samples at concentrations ranging from 100 to 4,500 pg/ml with no significant differences among the three groups. Control vitreous possessed the greatest FPA when compared with vitreous from eyes with PVR (p = 0.031) or RD (p = 0.048). These observations provide further evidence that cytokine-mediated pathways of inflammation are involved in the pathogenesis of PVR and point to the possible involvement of <em>IL</em>-1, <em>IL</em>-6 and IFN gamma in cellular interactions leading to chronicity.

How lymphocyte-mediated metal sensitivity affects orthopaedic implant performance remains poorly understood. Do patients with implants exhibit elevated lymphocyte reactivity to metals and is this reactivity more generalized or more implant-alloy specific? We investigated these questions by measuring lymphocyte responses to implant metals (Cr(+3), Co(+2), Ni(+2) at 0.1mM, and Ti(+4) at 0.001 mM) in six subject groups: Group 1a=young controls, Group 1b=age matched controls, Group 2a=subjects with osteoarthritis (OA) and no history of metal sensitivity, Group 2b=OA subjects with history of metal sensitivity, Group 3a=total hip arthroplasty (THA) subjects with no to mild radiographic osteolysis, and Group 3b=THA subjects with moderate osteolysis. Lymphocyte proliferation, using Lymphocyte Transformation Testing (LTT), and cytokine release provided quantitative reactivity measurement, where a stimulation index of >2 indicated metal sensitivity. OA subjects with a history of metal sensitivity (Group 2b) were more metal reactive to Ni than any other group, as expected (66% incidence and Stimulation Index>><em>20</em>). However, THA subjects (Groups 3a and b) were >3 fold more reactive to Cr (p<0.04), than were controls (Groups 1a & b) or OA subjects (Groups 2a & b). THA subjects with moderate vs mild osteolysis (Group 3b vs 3a) were more reactive to Co (43% vs 0% incidence). Only osteolytic THA subjects demonstrated increased cytokine responses with>>two-fold (p<0.05) increases in soluble interferon-gamma (IFN-gamma) and interleukin-2 (<em>IL</em>-2) levels in response to Cr challenge. This elevated incidence and averaged level of lymphocyte reactivity supports a metal-specific adaptive immune response and suggests involvement in the pathogenesis of poor implant performance, e.g. aseptic osteolysis.

Microvascular endothelial cells (EC) recruit circulating leukocytes at sites of inflammation, in part through cytokine-regulated expression of endothelial-leukocyte adhesion molecules. Adhesion molecule expression varies among vascular beds and among EC within microvessels of a particular vascular bed. In the present study, we have examined the patterns of antigen expression and cytokine responsiveness of dermal microvascular endothelial cells (DMEC) in a skin organ culture model and, for comparison, in cell culture. Within the superficial vascular plexus (SVP) of normal skin, CD36 molecule expression is undetectable on capillary loops and is expressed on DMEC in only <em>20</em>% of the larger, horizontal vessels. CD36 expression is not modulated by cytokines. Endothelial-leukocyte adhesion molecule-1 (ELAM-1) expression induced at 6 and 24 h by TNF or <em>IL</em>-1, is restricted to the venular side of the capillary loop and to the venules proper. Vascular cell adhesion molecule-1 (VCAM-1) expression is not inducible on EC of the SVP in normal skin by TNF, <em>IL</em>-1, or <em>IL</em>-4, alone or in combination at either time point. When inflamed skin is examined in organ culture, SVP EC are cytokine responsive regarding VCAM-1 expression. Within the deep vascular plexus (DVP). CD36 molecules are expressed on EC in all capillaries and small vessels. Both ELAM-1 and, to a lesser extent, VCAM-1 expression are inducible by TNF, <em>IL</em>-1, and/or <em>IL</em>-4 on capillaries and larger microvessels at 6 and 24 h. The larger vessels at the dermal-subcutaneous border were found to be CD36-/ELAM-1+/VCAM-1+ after cytokine treatment. CD36 expression of DMEC in cell culture varies from 47 to 98% of cells (mean 75%) in seven separate isolates and is not modified by cytokines. Upon TNF or <em>IL</em>-1 activation, 50 to 90% of DMEC express ELAM-1 molecules at 6 h and expression persists at high levels for 24 h. VCAM-1 expression is negligible at both times. These results with DMEC differ from human umbilical vein EC analyzed in parallel, which are completely CD36- and show transient ELAM-1 and sustained VCAM-1 expression in response to TNF and <em>IL</em>-1. In summary, we have demonstrated that DMEC comprise a heterogeneous population that differ from umbilical vein EC.