Single chain Fv chimeric receptors, or T-bodies, are described with intracellular sequences comprising the costimulatory signaling domain of CD28 in series with the zeta-chain from the TCR complex. Using an engineered human single chain Fv derived from P67, an mAb with specificity for human CD33, and a spacer comprising an Ab hinge region with either Fcgamma or part of the CD28 extracellular region, fusion molecules were constructed to test the ability of single chain designs to mediate both primary signaling and costimulation from one extracellular binding event. Constructs with the CD28 signaling domain proximal and the zeta-chain distal to the membrane were found to express more efficiently in Jurkat than constructs with the opposite orientation and were capable of mediating up to <em>20</em> times more <em>IL</em>-2 production on stimulation with solid phase Ag when compared with transfectants expressing chimeric receptors with zeta-chain intracellular signaling domains only. <em>IL</em>-2 production was specific to Ag challenge and was completely inhibited by incubation with free Ab of the same specificity as the extracellular binding site of the construct, but not by an isotype-matched control Ab. The CD28 intracellular domain of these fusion proteins was shown to be capable of binding the p85 subunit of phosphatidylinositol 3'-kinase. These constructs represent the first of a new generation of single gene multidomain chimeric receptors capable of mediating both primary and costimulatory signaling specifically from a single extracellular recognition event.

A structural, profile-based algorithm was used to identify interleukin <em>20</em> (<em>IL</em>-<em>20</em>), a novel <em>IL</em>-10 homolog. Chromosomal localization of <em>IL</em>-<em>20</em> led to the discovery of an <em>IL</em>-10 family cytokine cluster. Overexpression of <em>IL</em>-<em>20</em> in transgenic (TG) mice causes neonatal lethality with skin abnormalities including aberrant epidermal differentiation. Recombinant <em>IL</em>-<em>20</em> protein stimulates a signal transduction pathway through STAT3 in a keratinocyte cell line, demonstrating a direct action of this ligand. An <em>IL</em>-<em>20</em> receptor was identified as a heterodimer of two orphan class II cytokine receptor subunits. Both receptor subunits are expressed in skin and are dramatically upregulated in psoriatic skin. Taken together, these results demonstrate a role in epidermal function and psoriasis for <em>IL</em>-<em>20</em>, a novel cytokine identified solely by bioinformatics analysis.

Altered patterns of DNA methylation associated with Helicobacter pylori (HP) infection of gastric epithelial cells are thought to contribute to gastric cancer risk. However, it is unclear whether this increased risk reflects an infection-associated inflammatory response or the infection itself. In this study, we sought to clarify mechanisms in a gerbil model of gastric cancer where we showed that HP infection is causally involved in induction of aberrant DNA methylation. By genome-wide screening, CpG islands that were aberrantly methylated in gerbil gastric cancer cell lines were isolated, and 10 islands were shown to be specifically methylated only in gastric mucosae infected with HP. By temporal analysis, methylation levels in gastric epithelial cells started to increase at 5 to 10 weeks after infection and reached high levels by 50 weeks. When HP was eradicated, methylation levels markedly decreased 10 and <em>20</em> weeks later, but they remained higher than those in gerbils that were not infected by HP. Expression levels of several inflammation-related genes (CXCL2, <em>IL</em>-1beta, NOS2, and TNF-alpha) paralleled the temporal changes of methylation levels. Significantly suppressing inflammation with the immunosuppressive drug cyclosporin A did not affect colonization by HP but blocked the induction of altered DNA methylation. Our findings argue that DNA methylation alterations that occur in gastric mucosae after HP infection are composed of transient components and permanent components, and that it is the infection-associated inflammatory response, rather than HP itself, which is responsible for inducing the altered DNA methylation.

OBJECTIVE

To evaluate the safety and clinical efficacy of administering an anti-interleukin-10 (anti-IL-10) monoclonal antibody (mAb) to systemic lupus erythematosus (SLE) patients with active and steroid-dependent disease. In addition, we sought to assess the effects of in vivo IL-10 neutralization on biologic markers of SLE.

METHODS

Treatment consisted of 20 mg/day intravenous administration of an anti-IL-10 murine mAb (B-N10) for 21 consecutive days, with a followup period of 6 months. Six patients were studied.

RESULTS

Treatment was safe and well tolerated. All patients developed antibodies against B-N10. Cutaneous lesions and joint symptoms improved in all patients beginning during B-N10 administration and continuing to month 6. The SLE Disease Activity Index decreased from a mean +/- SEM of 8.83+/-0.91 on day 1 to 3.67+/-0.67 on day 21 (P = 0.001), 1.50+/-0.84 at month 2, and 1.33+/-0.80 at month 6 (P<0.001). At the end of followup, the disease was clinically inactive in 5 of the 6 patients. Prednisone administration was decreased from a mean +/- SEM of 27.9+/-5.7 mg/day on day 1 to 9.6+/-2.0 mg/day at month 6 (P<0.005). Activity of immune and endothelial cells rapidly decreased, as assessed by the early evolution of several biologic markers.

CONCLUSIONS

This is the first report of IL-10 antagonist administration to humans. The study shows the involvement of IL-10 in the pathogenesis of SLE, and indicates that the use of IL-10 antagonists may be beneficial in the management of refractory SLE.

Type 2 diabetes is associated with increased circulating concentrations of markers of the acute-phase response and interleukin-6 (<em>IL</em>-6). An augmented acute-phase response may be a mechanism which explains many of the clinical and biochemical features of type 2 diabetes and its complications. We sought to confirm that circulating concentrations of the cytokine acute-phase mediators <em>IL</em>-6 and tumour necrosis factor alpha [TNFalpha] are elevated in type 2 diabetes, and investigated blood as a source of cytokines in type 2 diabetes. Blood samples from <em>20</em> type 2 diabetic and 17 age-matched healthy subjects were incubated in vitro for 24 hr with and without lipopolysaccharide (LPS) stimulation and secreted cytokines measured. Plasma <em>IL</em>-6 and TNFalpha were significantly increased in type 2 diabetes compared to normal subjects. However, basal production of <em>IL</em>-6 and TNFalpha in cultured diabetic blood was markedly depressed in comparison with non-diabetic samples. <em>IL</em>-6 and TNFalpha production was increased in blood in response to LPS, reaching similar levels in diabetic and non-diabetic subjects, though <em>IL</em>-6 was slightly but significantly higher in controls. We conclude that circulating levels of <em>IL</em>-6 and TNFalpha are increased in type 2 diabetes but there is downregulation of basal cytokine production in blood cells in type 2 diabetes. Blood has the capacity to produce cytokines in diabetes which contribute to the augmented acute-phase response, but the main source of the increased plasma <em>IL</em>-6 and TNFalpha concentrations may be from non-circulating cells.

<em>IL</em>-1alpha, like <em>IL</em>-1beta, possesses multiple inflammatory and immune properties. However, unlike <em>IL</em>-1beta, the cytokine is present intracellularly in healthy tissues and is not actively secreted. Rather, <em>IL</em>-1alpha translocates to the nucleus and participates in transcription. Here we show that intracellular <em>IL</em>-1alpha is a chromatin-associated cytokine and highly dynamic in the nucleus of living cells. During apoptosis, <em>IL</em>-1alpha concentrates in dense nuclear foci, which markedly reduces its mobile nature. In apoptotic cells, <em>IL</em>-1alpha is retained within the chromatin fraction and is not released along with the cytoplasmic contents. To simulate the in vivo inflammatory response to cells undergoing different mechanisms of death, lysates of cells were embedded in Matrigel plugs and implanted into mice. Lysates from cells undergoing necrosis recruited cells of the myeloid lineage into the Matrigel, whereas lysates of necrotic cells lacking <em>IL</em>-1alpha failed to recruit an infiltrate. In contrast, lysates of cells undergoing apoptotic death were inactive. Cells infiltrating the Matrigel were due to low concentrations (<em>20</em>-50 pg) of the <em>IL</em>-1alpha precursor containing the receptor interacting C-terminal, whereas the N-terminal propiece containing the nuclear localization site failed to do so. When normal keratinocytes were subjected to hypoxia, the constitutive <em>IL</em>-1alpha precursor was released into the supernatant. Thus, after an ischemic event, the <em>IL</em>-1alpha precursor is released by hypoxic cells and incites an inflammatory response by recruiting myeloid cells into the area. Tissues surrounding the necrotic site also sustain damage from the myeloid cells. Nuclear trafficking and differential release during necrosis vs. apoptosis demonstrate that inflammation by <em>IL</em>-1alpha is tightly controlled.

Oral tolerance was generated to hen egg white lysozyme in the mouse or to guinea pig myelin basic protein in the rat by a low-dose (1 mg) or a high-dose (5-<em>20</em> mg) feeding regimen. High doses of antigen induced tolerance characterized by anergy with little or no active suppression and increased secretion of interleukin 4 (<em>IL</em>-4). Anergy was shown by an increase in frequency of <em>IL</em>-2-secreting cells following culture in recombinant <em>IL</em>-2. Low doses of antigen induced tolerance characterized by antigen-driven active suppression with increased secretion of transforming growth factor beta (TGF-beta) and <em>IL</em>-4 and minimal anergy. Without further immunization, spleen cells from animals orally tolerized by both regimens secreted increased levels of <em>IL</em>-4 and TGF-beta in an antigen-specific manner. Animals fed high doses secreted more <em>IL</em>-4 and less TGF-beta, whereas those fed low doses secreted more TGF-beta and less <em>IL</em>-4. These results demonstrate that the two feeding regimens induced cell populations that differed in their cytokine secretion profile and their capacity to actively suppress in vitro and to induce anergy. Our results provide a basis for distinguishing different forms of antigen-driven peripheral tolerance and have important implications for orally induced antigen-specific modulation of human autoimmune diseases.

Several chemotactic agonists including interleukin-8 (<em>IL</em>-8) and related cytokines have been shown to activate and attract leukocytes via seven-transmembrane domain, GTP-binding protein-coupled receptors. A cDNA clone, LESTR, encoding a protein of 352 amino acids, corresponding to a novel receptor of this type, was isolated from a human blood monocyte cDNA library. The sequence of the deduced protein, LESTR (leukocyte-derived seven-transmembrane domain receptor), has 92.6% identity with that of a recently reported bovine neuropeptide Y (NPY) receptor, boLCR1 (Rimland, J., Xin, W., Sweetnam, P., Saijoh, K., Nestler, E. J., and Duman, R. S. (1991) Mol. Pharmacol. 40, 869-875). LESTR, however, is more similar >> 34%) to the <em>IL</em>-8 receptors, <em>IL</em>-8R1 and <em>IL</em>-8R2, than to several NPY receptors of different origin (< <em>20</em>%). In the monocyte library, LESTR cDNA fragments were about <em>20</em> times as frequent as cDNA coding for <em>IL</em>-8R1 and <em>IL</em>-8R2, and much higher levels of LESTR- than <em>IL</em>-8R-specific mRNA were found in human blood neutrophils and lymphocytes. LESTR transcripts, by contrast, were low or undetectable in several neuroblastoma cell lines that are widely used to study NPY functions. Transfected cells expressing high levels of LESTR mRNA did not bind radiolabeled NPY, <em>IL</em>-8, NAP-2, GRO alpha, PF4, IP10, MCP-1, MCP-3, MIP-1 alpha, HC14, I309, RANTES, C3a, or LTB4. NPY also failed to bind to neutrophils, monocytes, and lymphocytes, to elicit responses in vitro such as Ca2+ changes, shape change, chemotaxis, enzyme release, and the respiratory burst, and to induce leukocyte accumulation upon injection in rats and rabbits. Although the ligand for LESTR could not be identified among a large number of chemotactic cytokines, the high expression in white blood cells and the marked sequence relation to <em>IL</em>-8R1 and <em>IL</em>-8R2 suggest that LESTR may function in the activation of inflammatory cells.

BACKGROUND

Nasal polyps (NPs) are characterized by eosinophilic inflammation and often coexist with asthma. However, the role of atopy and IgE in NP pathogenesis is unclear.

OBJECTIVE

We sought to determine whether there is an association between total and specific IgE to a variety of allergens in polyp and nonpolyp tissue and markers of eosinophilic inflammation or skin test results.

METHODS

Homogenates were prepared from nasal tissue of <em>20</em> patients with NPs and <em>20</em> patients without NPs and analyzed for concentrations of <em>IL</em>-5, <em>IL</em>-4, eotaxin, leukotriene (LT) C4/D4/E4, sCD23, and histamine (ELISA). Eosinophil cationic protein (ECP), tryptase, and total and specific IgE for inhalant allergens and Staphylococcus aureus enterotoxins were measured (ImmunoCAP).

RESULTS

The concentrations of total IgE, IL-5, eotaxin, ECP, LTC4/D4/E4, and sCD23 were significantly higher in NP tissue compared with nonpolyp tissue. Total IgE was significantly correlated to IL-5, ECP, LTC4/D4/E4, and sCD23 and to the number of eosinophils in NPs. On the basis of the presence of specific IgE antibodies in tissue, 3 NP groups were defined. NP group 1 demonstrated no measurable specific IgE, and NP group 2 selected specific IgE. The third group demonstrated a multiclonal specific IgE, including IgE to S aureus enterotoxins, a high total IgE level, and a high prevalence of asthma.

CONCLUSIONS

These studies suggest that there is an association between increased levels of total IgE, specific IgE, and eosinophilic inflammation in NPs, which may be of relevance in the pathophysiology of nasal polyposis. Similarly, the presence of specific IgE to staphylococcal enterotoxins A and B also points to a possible role of bacterial superantigens.

T helper (Th) 17 cells have recently been implicated in psoriasis pathogenesis, but mechanisms of how these cells traffic into inflamed skin are unknown. By immunostaining for interleukin (<em>IL</em>)-17A and <em>IL</em>-22, we show numerous cells present in psoriasis lesions that produce these cytokines. We next found that Th17 cytokines (<em>IL</em>-17A, <em>IL</em>-22, and tumor necrosis factor (TNF)-alpha) markedly increased the expression of CC chemokine ligand (CCL) <em>20</em>, a CC chemokine receptor (CCR)6 ligand, in human keratinocyte monolayer and raft cultures in a dose- and time-dependent manner. Lastly, we showed in mice that subcutaneous injection with recombinant <em>IL</em>-17A, <em>IL</em>-22, or TNF-alpha led to the upregulation of both CCL<em>20</em> and CCR6 expression in skin as well as cutaneous T-cell infiltration. Taken together, these data show that Th17 cytokines stimulate CCL<em>20</em> production in vitro and in vivo, and thus provide a potential explanation of how CCR6-positive Th17 cells maintain their continual presence in psoriasis through a positive chemotactic feedback loop.

microRNAs are thought to regulate tumor progression and invasion via direct interaction with target genes within cells. Here the microRNA17/<em>20</em> cluster is shown to govern cellular migration and invasion of nearby cells via heterotypic secreted signals. microRNA17/<em>20</em> abundance is reduced in highly invasive breast cancer cell lines and node-positive breast cancer specimens. Cell-conditioned medium from microRNA17/<em>20</em>-overexpressing noninvasive breast cancer cell MCF7 was sufficient to inhibit MDA-MB-231 cell migration and invasion through inhibiting secretion of a subset of cytokines, and suppressing plasminogen activation via inhibition of the secreted plasminogen activators (cytokeratin 8 and alpha-enolase). microRNA17/<em>20</em> directly repressed <em>IL</em>-8 by targeting its 3' UTR, and inhibited cytokeratin 8 via the cell cycle control protein cyclin D1. At variance with prior studies, these results demonstrated a unique mechanism of how the altered microRNA17/<em>20</em> expression regulates cellular secretion and tumor microenvironment to control migration and invasion of neighboring cells in breast cancer. These findings not only reveal an antiinvasive function of miR-17/<em>20</em> in breast cancer, but also identify a heterotypic secreted signal that mediates the microRNA regulation of tumor metastasis.

OBJECTIVE

Current biomarkers cannot completely distinguish sepsis from systemic inflammatory response syndrome (SIRS) caused by other non-infectious diseases. Circulating microRNAs (miRNAs) are promising biomarkers for several diseases, but their correlation with sepsis is not totally clarified.

METHODS

Seven miRNAs related to inflammation or infection were included in the present study. Serum miRNA expression was investigated in 50 patients diagnosed with sepsis, 30 patients with SIRS and <em>20</em> healthy controls to evaluate the diagnostic and prognostic value. Expression levels of serum miRNAs were determined by quantitative PCR using the Qiagen miScript system. Serum CRP and <em>IL</em>-6 levels were determined by enzyme linked immunosorbent assay.

RESULTS

Serum miR-146a and miR-223 were significantly reduced in septic patients compared with SIRS patients and healthy controls. The areas under the receiver operating characteristic curve of miR-146a, miR-223 and IL-6 were 0.858, 0.804 and 0.785, respectively.

CONCLUSIONS

Serum miR-146a and miR-223 might serve as new biomarkers for sepsis with high specificity and sensitivity. (ClinicalTrials.gov number, NCT00862290.).

It is unknown why only a minority of circulating tumor cells trapped in lung capillaries form metastases and involvement of immune cells remains uncertain. A novel model has been developed in this study showing that neutrophils regulate lung metastasis development through physical interaction and anchoring of circulating tumor cells to endothelium. Human melanoma cells were i.v. injected into nude mice leading to the entrapment of many cancer cells; however, 24 hours later, very few remained in the lungs. In contrast, injection of human neutrophils an hour after tumor cell injection increased cancer cell retention by approximately 3-fold. Entrapped melanoma cells produced and secreted high levels of a cytokine called interleukin-8 (<em>IL</em>-8), attracting neutrophils and increasing tethering beta(2) integrin expression by 75% to 100%. Intercellular adhesion molecule-1 on melanoma cells and beta(2) integrin on neutrophils interacted, promoting anchoring to vascular endothelium. Decreasing <em>IL</em>-8 secretion from melanoma cells lowered extracellular levels by <em>20</em>% to 50%, decreased beta(2) integrin on neutrophils by approximately 50%, and reduced neutrophil-mediated extravasation by 25% to 60%, resulting in approximately 50% fewer melanoma cells being tethered to endothelium and retained in lungs. Thus, transendothelial migration and lung metastasis development decreased by approximately 50%, showing that targeting <em>IL</em>-8 in melanoma cells has the potential to decrease metastasis development by disrupting interaction with neutrophils.

Erlotinib (Tarceva) is an orally available HER1 (epidermal growth factor receptor, EGFR) tyrosine kinase inhibitor advancing through clinical trials for the treatment of a range of human malignancies. In this study, we examine the capacity of erlotinib to modulate radiation response and investigate specific mechanisms underlying these interactions in human tumor cell lines and xenografts. The impact of erlotinib on cell cycle kinetics was analyzed using flow cytometry, and the impact on apoptosis was evaluated via fluorescein-labeled pan-caspase inhibition and poly(ADP-ribose) polymerase cleavage. Radiation-induced EGFR autophosphorylation and Rad51 expression were examined by Western blot analysis. Radiation survival was analyzed using a clonogenic assay and assessment of in vivo tumor growth was done using a mouse xenograft model system. Microarray studies were carried out using <em>20</em> K human cDNA microarray and select genes were validated using quantitative reverse transcription-PCR (RT-PCR). Independently, erlotinib and radiation induce accumulation of tumor cells in G(1) and G(2)-M phase, respectively, with a reduction of cells in S phase. When combined with radiation, erlotinib promotes a further reduction in S-phase fraction. Erlotinib enhances the induction of apoptosis, inhibits EGFR autophosphorylation and Rad51 expression following radiation exposure, and promotes an increase in radiosensitivity. Tumor xenograft studies confirm that systemic administration of erlotinib results in profound tumor growth inhibition when combined with radiation. cDNA microarray analysis assessing genes differentially regulated by erlotinib following radiation exposure identifies a diverse set of genes deriving from several functional classes. Validation is confirmed for several specific genes that may influence radiosensitization by erlotinib including Egr-1, CXCL1, and <em>IL</em>-1beta. These results identify the capacity of erlotinib to enhance radiation response at several levels, including cell cycle arrest, apoptosis induction, accelerated cellular repopulation, and DNA damage repair. Preliminary microarray data suggests additional mechanisms underlying the complex interaction between EGFR signaling and radiation response. These data suggest that the erlotinib/radiation combination represents a strategy worthy of further examination in clinical trials.

OBJECTIVE

Recent studies suggest that exchange of macrophage phenotype (M1/M2) in adipose tissue is associated with chronic low-grade inflammation in obesity. M1 macrophages enhance a chronic inflammatory state in adipose tissues, whereas M2 macrophages inhibit it. Although exercise training might inhibit pro-inflammatory cytokine gene expression in adipose tissue, it remains unclear whether exercise training affects the phenotypic switch of macrophage polarization in adipose tissue. Therefore, we inveStigated the effect of exercise training on the macrophage phenotypic switch in adipose tissue in high-fat-induced obese mice.

METHODS

Male C57BL/6 mice were divided into four groups; normal diet (ND) control (n=7), ND exercise (n=7), high-fat-diet (HFD) control (n=12), and HFD exercise (n=12) groups. All exercised mice ran on a treadmill at 12-<em>20</em> m/min for 60 min/day for 16 weeks. Tumor necrosis factor (TNF)-alpha, interleukin (<em>IL</em>)-6, F4/80, monocyte chemotactic protein (MCP)-1, CXCL14, inter-cellular adhesion molecule (ICAM)-1, vascular-cellular adhesion molecule (VCAM)-1, CD11c, CD163 and toll-like receptor (TLR)4 mRNA expressions in adipose tissue were evaluated by real time-RT-PCR.

RESULTS

In HFD mice, exercise training did not induce loss of body or adipose tissue mass, exercise training nevertheless markedly inhibited TNF-alpha and F4/80 mRNA expression in adipose tissue. The exercise training attenuated HFD-induced increase in ICAM-1 mRNA expression, but not MCP-1, CXCL14 and VCAM-1 mRNA expressions. In addition, increased CD11c mRNA expression, which is a M1 macrophage specific marker, with HFD treatment was attenuated by exercise training. In contrast, although the mRNA expression of CD163, a M2 macrophage specific marker, in adipose tissue was significantly decreased by HFD, the exercise training significantly increased its expression. Also, the higher mRNA expression of TLR4, which induces pro-inflammatory cytokine production after fatty acid recognition, was strongly inhibited by the exercise training in HFD mice.

CONCLUSIONS

Exercise training might induce the phenotypic switching from M1 macrophage to M2 macrophage in obese adipose tissue besides inhibiting M1 macrophage infiltration into adipose tissue. Therefore, chronic exercise might contribute to inhibit inflammation in adipose tissue via down regulation of TLR4.

The Gfi-1 proto-oncogene is activated by provirus insertion in T-cell lymphoma lines selected for interleukin-2 (<em>IL</em>-2) independence in culture and in primary retrovirus-induced thymomas and encodes a nuclear, sequence-specific DNA-binding protein. Here we show that Gfi-1 is a position- and orientation-independent active transcriptional repressor, whose activity depends on a <em>20</em>-amino-acid N-terminal repressor domain, coincident with a nuclear localization motif. The sequence of the Gfi-1 repressor domain is related to the sequence of the repressor domain of Gfi-1B, a Gfi-1-related protein, and to sequences at the N termini of the insulinoma-associated protein, IA-1, the homeobox protein Gsh-1, and the vertebrate but not the Drosophila members of the Snail-Slug protein family (Snail/Gfi-1, SNAG domain). Although not functionally characterized, these SNAG-related sequences are also likely to mediate transcriptional repression. Therefore, the Gfi-1 SNAG domain may be the prototype of a novel family of evolutionarily conserved repressor domains that operate in multiple cell lineages. Gfi-1 overexpression in <em>IL</em>-2-dependent T-cell lines allows the cells to escape from the G1 arrest induced by <em>IL</em>-2 withdrawal. Since a single point mutation in the SNAG domain (P2A) inhibits both the Gfi-1-mediated transcriptional repression and the G1 arrest induced by <em>IL</em>-2 starvation, we conclude that the latter depends on the repressor activity of the SNAG domain. Induction of Gfi-1 may therefore contribute to T-cell activation and tumor progression by repressing the expression of genes that inhibit cellular proliferation.

We tested the hypothesis that, during sepsis, the balance of pro- and anti-inflammatory cytokines is related to severity and survival. Cecal ligation and puncture (CLP) with a large (18-gauge)-, intermediate (21-gauge)-, or small (26-gauge)-diameter needle, or sham laparotomy, was performed on outbred CD-1 mice. Concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (<em>IL</em>-6), and the anti-inflammatory cytokine <em>IL</em>-10 were measured (by enzyme-linked immunosorbent assay) in serum, peritoneal lavage fluid, and liver and lung samples at 4, 8, 24, 48, and 96 h. As the diameter of the CLP needle decreased, the mortality rate decreased (at 48 h: large, 80%; intermediate, 40%; small, <em>20</em>%; P < 0.05), the TNF-alpha and <em>IL</em>-6 concentrations decreased, and the time-to-peak TNF-alpha expression increased. In contrast, <em>IL</em>-10 concentration increased compared with baseline (serum at 24 h: large, 2.3-fold +/- 1.6-fold; intermediate, 2.0-fold +/- 0.5-fold; small, 49.9-fold +/- 8.3-fold; P < 0.05). Administration of <em>IL</em>-10 (5 microg, intraperitoneal) prior to CLP decreased mortality (P < 0.001). Administration of polyclonal anti-<em>IL</em>-10 serum prior to CLP (0.5 ml intraperitoneal) had the opposite effect and increased mortality (P < 0.001) and TNF-alpha, <em>IL</em>-6, and TNF-alpha mRNA expression compared with controls. Thus, severe sepsis is associated with a largely unopposed inflammatory response, and a largely unopposed inflammatory response (with anti-<em>IL</em>-10) results in severe sepsis and death. Less severe sepsis is associated with greater anti-inflammatory mediator expression, and greater anti-inflammatory mediator expression (with <em>IL</em>-10) results in less severe sepsis. Thus, the balance of inflammatory mediators is related to the severity and mortality of murine sepsis.

The aspartate-specific cysteine protease caspase-1 is activated by the inflammasomes and is responsible for the proteolytic maturation of the cytokines <em>IL</em>-1 beta and <em>IL</em>-18 during infection and inflammation. To discover new caspase-1 substrates, we made use of a proteome-wide gel-free differential peptide sorting methodology that allows unambiguous localization of the processing site in addition to identification of the substrate. Of the 1022 proteins that were identified, <em>20</em> were found to be specifically cleaved after Asp in the setup incubated with recombinant caspase-1. Interestingly, caspase-7 emerged as one of the identified caspase-1 substrates. Moreover half of the other identified cleavage events occurred at sites closely resembling the consensus caspase-7 recognition sequence DEVD, suggesting caspase-1-mediated activation of endogenous caspase-7 in this setup. Consistently recombinant caspase-1 cleaved caspase-7 at the canonical activation sites Asp(23) and Asp(198), and recombinant caspase-7 processed a subset of the identified substrates. In vivo, caspase-7 activation was observed in conditions known to induce activation of caspase-1, including Salmonella infection and microbial stimuli combined with ATP. Interestingly Salmonella- and lipopolysaccharide + ATP-induced activation of caspase-7 was abolished in macrophages deficient in caspase-1, the pattern recognition receptors Ipaf and Cryopyrin, and the inflammasome adaptor ASC, demonstrating an upstream role for the caspase-1 inflammasomes in caspase-7 activation in vivo. In contrast, caspase-1 and the inflammasomes were not required for caspase-3 activation. In conclusion, we identified <em>20</em> new substrates activated downstream of caspase-1 and validated caspase-1-mediated caspase-7 activation in vitro and in knock-out macrophages. These results demonstrate for the first time the existence of a nucleotide binding and oligomerization domain-like receptor/caspase-1/caspase-7 cascade and the existence of distinct activation mechanisms for caspase-3 and -7 in response to microbial stimuli and bacterial infection.

A panning method has been developed to enrich Langerhans cells (LC) from murine epidermis. In standard culture media, the enriched populations progressively lose viability over a 3-d interval. When the cultures are supplemented with keratinocyte-conditioned medium, LC viability is improved and the cells increase in size and number of dendritic processes. Accessory function, as monitored by stimulating activity in the mixed lymphocyte reaction (MLR), increases at least 10-<em>20</em>-fold. The conditioned media of stimulated macrophages and T cells also support the viability and maturation of cultured LC. A panel of purified cytokines has been tested, and only granulocyte/macrophage colony-stimulating factor (GM-CSF) substitutes for bulk-conditioned medium. The recombinant molecule exhibits half-maximal activity at 5 pM. Without activity are: <em>IL</em>-1-4; IFN-alpha/beta/gamma; cachectin/TNF; M- and G-CSF. A rabbit anti-GM-CSF specifically neutralizes the capacity of keratinocyte-conditioned medium to generate active LC. However, GM-CSF is not required for LC function during the MLR itself. We conclude that the development of immunologically active LC in culture is mediated by GM-CSF. The observation that these dendritic cells do not respond to lineage-specific G- and M-CSFs suggests that LC represent a distinct myeloid differentiation pathway. Because GM-CSF can be made by nonimmune cells and can mediate the production of active dendritic cells, this cytokine provides a T-independent mechanism for enhancing the sensitization phase of cell-mediated immunity.

Leukocytes and vascular cells interact closely in inflammation and immunity and lymphokines are important mediators of this interaction. The present study was designed to define the possible role of <em>IL</em>-6 as a communication signal between vascular and immunocompetent cells. <em>IL</em>-6 was measured as hybridoma growth factor (HGF) on the 7TD1 cell line in the supernatants of human endothelial cells (HEC). HEC released appreciable levels of HGF activity in the absence of deliberate stimulation. In vitro exposure to recombinant <em>IL</em>-1 beta markedly increased (usually 10 to 15-fold) HGF production by HEC. Optimal stimulation was observed with 0.1 to 50 U/ml for 4 to <em>20</em> h of incubation. Human and murine r<em>IL</em>-1 alpha stimulated HGF production in HEC. Anti-<em>IL</em>-6 antibodies inhibited the HGF activity of the HEC supernatants, thus confirming, together with the cytokine specificity of the assay, the nature of HEC-produced cytokine. <em>IL</em>-1-treated HEC expressed high levels of <em>IL</em>-6 mRNA as detected by Northern blot analysis. Inasmuch as <em>IL</em>-1 elicits a complex series of changes in HEC, it was important to assess whether <em>IL</em>-6, produced after exposure to <em>IL</em>-1, modified HEC function. Natural or r<em>IL</em>-6 did not affect the functional status of HEC as assessed by proliferative capacity, production of procoagulant activity and prostacyclin, ability to induce adhesion of polymorphonuclear leukocytes. The capacity to produce <em>IL</em>-6 may represent an important mechanism by which endothelial cells participate in inflammatory and immune reactions.

The role of monocytes/macrophages in the pathogenesis of ischemia-reperfusion injury (IRI) is unknown. We sought to determine whether activation of macrophage adenosine 2A (A(2A)) receptors (A(2A)Rs) mediates tissue protection. We subjected C57Bl/6 mice infused with clodronate [dichloromethylene bisphosphonate (Cl(2)MBP)] to IRI (32 min of ischemia followed by 24 h of reperfusion) to deplete them of macrophages. IRI induced an elevation of plasma creatinine that was reduced with Cl(2)MBP (26% of control). Adoptive transfer of murine RAW 264.7 cells reconstituted injury, an effect blocked significantly by A(2A) agonists (27% of plasma creatinine from mice reconstituted with macrophages). Macrophages subjected to A(2A) knockout by small interfering RNA were adoptively transferred to macrophage-depleted mice and reconstituted injury (110% of control mice); however, the increase in plasma creatinine was blocked by A(2A) agonists (<em>20</em>% of vehicle treatment). Finally, the A(2A) agonist effect on IRI was blocked in macrophage-depleted A(2A)-knockout mice reconstituted with wild-type RAW 264.7 cells. RNase protection assays 24 h after IRI demonstrated that macrophages are required for <em>IL</em>-6 and TGF-beta mRNA induction. However, A(2A) agonist-mediated tissue protection is independent of <em>IL</em>-6 and TGF-beta mRNA. We conclude that the full extent of IRI requires macrophages and that A(2A) agonist-mediated tissue protection is independent of activation of macrophage A(2A)Rs.

Mucosal changes in inflammatory bowel disease (IBD) are characterized by ulcerative lesions accompanied by prominent cellular infiltrates in the bowel wall. Chemokines are chemotactic cytokines that are able to promote leukocyte migration to areas of inflammation and are also able to initiate cell activation events. They have recently been implicated in the pathophysiology of many disease states. The aim of this study was to detail the degree and distribution of specific chemokines, interleukin (<em>IL</em>)-8, monocyte chemoattractant protein (MCP)-1, -2, and -3, and macrophage inflammatory protein (MIP)-1alpha and -1beta, in IBD mucosa. Thirty-nine patients were included, ten controls, <em>20</em> ulcerative colitis (UC), and nine Crohn's disease (CD), with a range of disease activity. Colonic mucosal biopsies were collected from UC, CD, and control patients and embedded in glycol methacrylate. Two-micrometre-thick sections were cut and stained using immunohistochemistry for chemokine protein expression. Sections were analysed using a light microscope. Expression of all types of chemokine protein was detected in colonic mucosa from both control and IBD patients. Patterns of staining between IBD patients and controls differed significantly, but CD and UC patients demonstrated similar patterns of staining. Individual chemokine expression was found to be significantly up-regulated in IBD when patients were compared with the non-diseased group in all areas of the mucosal sections. Up-regulated chemokine expression correlated with increasing activity of the disease. It is concluded that human colonic chemokine expression is non-selectively up-regulated in IBD. The results supported the hypothesis that the degree of local inflammation and tissue damage in UC and CD is dependent on local expression of specific chemokines within IBD tissues.

Trans fatty acid intake has been associated with a higher risk of cardiovascular disease. The relation is explained only partially by the adverse effect of these fatty acids on the lipid profile. We examined whether trans fatty acid intake could also affect biomarkers of inflammation and endothelial dysfunction including C-reactive protein (CRP), interleukin-6 (<em>IL</em>-6), soluble tumor necrosis factor receptor 2 (sTNFR-2), E-selectin, and soluble cell adhesion molecules (sICAM-1 and sVCAM-1). We conducted a cross-sectional study of 730 women from the Nurses' Health Study I cohort, aged 43-69 y, free of cardiovascular disease, cancer, and diabetes at time of blood draw (1989-1990). Dietary intake was assessed by a validated FFQ in 1986 and 1990. CRP levels were 73% higher among those in the highest quintile of trans fat intake, compared with the lowest quintile. <em>IL</em>-6 levels were 17% higher, sTNFR-2 5%, E-selectin <em>20</em>%, sICAM-1 10%, and sVCAM-1 levels 10% higher. Trans fatty acid intake was positively related to plasma concentration of CRP (P = 0.009), sTNFR-2 (P = 0.002), E-selectin (P = 0.003), sICAM-1 (P = 0.007), and sVCAM-1 (P = 0.001) in linear regression models after controlling for age, BMI, physical activity, smoking status, alcohol consumption, intake of monounsaturated, polyunsaturated, and saturated fatty acids, and postmenopausal hormone therapy. In conclusion, this study suggests that higher intake of trans fatty acids could adversely affect endothelial function, which might partially explain why the positive relation between trans fat and cardiovascular risk is greater than one would predict based solely on its adverse effects on lipids.

OBJECTIVE

Renal cancer response to interleukin 2 (IL-2) therapy and patient survival has been correlated with tumor histology and carbonic anhydrase IX (CAIX) expression. In an effort to confirm and expand these observations, we examined CAIX expression in pathology specimens from renal cancer patients who had previously received IL-2 therapy.

METHODS

Paraffin-embedded tissue sections of renal cancer were immunostained with the MN-75 monoclonal antibody to CAIX and expression levels were correlated with histologic findings and clinical outcome.

RESULTS

Tissue specimens were obtained from 66 patients; 27 of whom (41%) had responded to IL-2-based therapy. Fifty-eight specimens were assessed as clear cell, with 56, 33, and 4 having alveolar, granular, and papillary features, respectively. Twenty-four (36%), 31 (47%), and 11 (17%) were classified into good, intermediate, and poor prognosis groups according to the Upton pathology model. Forty-one specimens (62%) had high CAIX expression. Twenty-one of 27 (78%) responding patients had high CAIX expressing tumors compared with 20 of 39 (51%) nonresponders (odds ratio, 3.3; P = 0.04). Median survival was prolonged (P = 0.04) and survival >5 years was only seen in high CAIX expressers. In patients with intermediate pathologic prognosis, all nine responders had high CAIX expression versus 11 of 22 nonresponders. A resultant group with good pathologic prognosis alone or with intermediate pathologic prognosis and high CAIX contained 26 of 27 (96%) responders compared with 18 of 39 (46%) nonresponders (odds ratio, 30; P < 0.01) and exhibited longer median survival (P < 0.01).

CONCLUSIONS

CAIX expression seems to be an important predictor of outcome in renal cell carcinoma patients receiving IL-2-based therapy and may enhance prognostic information obtained from pathology specimens.