OBJECTIVE

To analyze the frequency, surface phenotype, and cytokine secretion of CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) compared with both healthy control subjects and patients with rheumatoid arthritis (RA).

METHODS

Eight-color flow cytometry was used to analyze the surface phenotype and cytokine production of PBMCs from <em>20</em> patients with AS, 12 patients with RA, and 16 healthy control subjects, following stimulation ex vivo with phorbol myristate acetate and ionomycin for 5 hours. Secretion of interleukin-17 (<em>IL</em>-17) by PBMCs was measured by enzyme-linked immunosorbent assay, following stimulation with anti-CD3/CD28 for 4 days.

RESULTS

The percentages of IL-17-positive CD4+ T cells and IL-22-positive CD4+ T cells were increased in the PBMCs of both patients with AS and patients with RA compared with healthy control subjects, whereas there were no differences in the percentages of interferon-gamma (IFNgamma)-positive or IL-10-positive CD4+ T cells. Likewise, concentrations of IL-17 in supernatants from patients with AS were significantly higher compared with those from healthy control subjects. In patients with RA, the concentrations of IL-17 were increased but not significantly. There was a correlation between the percentages of IL-17-positive CD4+ T cells detected in PBMCs and the amounts of IL-17 in culture supernatants (r=0.414, P=0.0034). All IL-17-producing cells were CD4+CD45RO+; most expressed both CCR6 and CCR4, but only 50% expressed the IL-23 receptor (IL-23R). Nevertheless, there was a positive relationship between the percentage of IL-23R-positive CD4+ T cells and the frequency of IL-17-positive CD4+ T cells or IL-22-positive CD4+ T cells (r=0.57, P<0.0001 and r=0.46, P=0.001, respectively). A significant proportion of cells that produced IL-17 also produced IL-22 and IFNgamma, but none produced IL-10.

CONCLUSIONS

The frequencies of IL-17-positive and IL-22-positive CD4+ T cells were increased in PBMCs from patients with AS and patients with RA, resulting in secretion of higher quantities of IL-17 by PBMCs following stimulation. These data support the hypothesis that Th17 cells, particularly when present in excess of IL-10-producing cells, are involved in the pathogenesis of inflammatory arthritis.

Monocytes (Mo) and macrophages (MΦ) are emerging therapeutic targets in malignant, cardiovascular, and autoimmune disorders. Targeting of Mo/MΦ and their effector functions without compromising innate immunity's critical defense mechanisms first requires addressing gaps in knowledge about the life cycle of these cells. Here we studied the source, tissue kinetics, and clearance of Mo/MΦ in murine myocardial infarction, a model of acute inflammation after ischemic injury. We found that a) Mo tissue residence time was surprisingly short (<em>20</em> h); b) Mo recruitment rates were consistently high even days after initiation of inflammation; c) the sustained need of newly made Mo was fostered by extramedullary monocytopoiesis in the spleen; d) splenic monocytopoiesis was regulated by <em>IL</em>-1β; and e) the balance of cell recruitment and local death shifted during resolution of inflammation. Depending on the experimental approach, we measured a 24 h Mo/MΦ exit rate from infarct tissue between 5 and 13% of the tissue cell population. Exited cells were most numerous in the blood, liver, and spleen. Abrogation of extramedullary monocytopoiesis proved deleterious for infarct healing and accelerated the evolution of heart failure. We also detected rapid Mo kinetics in mice with stroke. These findings expand our knowledge of Mo/MΦ flux in acute inflammation and provide the groundwork for novel anti-inflammatory strategies for treating heart failure.

The role of LL-37, a human cationic antimicrobial peptide, in the immune system is not yet clearly understood. It is a widely expressed peptide that can be up-regulated during an immune response. In this report, we demonstrate that LL-37 is a potent antisepsis agent with the ability to inhibit macrophage stimulation by bacterial components such as LPS, lipoteichoic acid, and noncapped lipoarabinomannan. We also demonstrate that LL-37 protects mice against lethal endotoxemia. In addition to preventing macrophage activation by bacterial components, we hypothesized the LL-37 may also have direct effects on macrophage function. We therefore used gene expression profiling to identify macrophage functions that might be modulated by LL-37. These studies revealed that LL-37 directly up-regulates 29 genes and down-regulated another <em>20</em> genes. Among the genes predicted to be up-regulated by LL-37 were those encoding chemokines and chemokine receptors. Consistent with this, LL-37 up-regulated the expression of chemokines in macrophages and the mouse lung (monocyte chemoattractant protein 1), human A549 epithelial cells (<em>IL</em>-8), and whole human blood (monocyte chemoattractant protein 1 and <em>IL</em>-8), without stimulating the proinflammatory cytokine, TNFalpha. LL-37 also up-regulated the chemokine receptors CXCR-4, CCR2, and <em>IL</em>-8RB. These findings indicate that LL-37 may contribute to the immune response by limiting the damage caused by bacterial products and by recruiting immune cells to the site of infection so that they can clear the infection.

This study investigated the expression of five novel human <em>IL</em>-10-related molecules and their receptors in blood mononuclear cells. <em>IL</em>-19 and <em>IL</em>-<em>20</em> were found to be preferentially expressed in monocytes. <em>IL</em>-22 and <em>IL</em>-26 (AK155) expression was exclusively detected in T cells, especially upon type 1 polarization, and in NK cells. <em>IL</em>-24 (melanoma differentiation-associated gene 7) expression was restricted to monocytes and T cells. Detection of these molecules in lymphocytes was predominantly linked to cellular activation. Regarding T cells, <em>IL</em>-26 was primarily produced by memory cells, and its expression was independent on costimulation. In contrast to the high expression of receptors for <em>IL</em>-10 homologs in different tissues and cell lines, monocytes and NK, B, and T cells showed clear expression only of <em>IL</em>-10R1, <em>IL</em>-10R2, and <em>IL</em>-<em>20</em>R2. In these cells, <em>IL</em>-<em>20</em>R2 might be part of a still-unknown receptor complex. Therefore, immune cells may represent a major source but a minor target of the novel <em>IL</em>-10 family members.

BACKGROUND

Patients with chronic obstructive pulmonary disease (COPD) are prone to frequent exacerbations which are a significant cause of morbidity and mortality. Stable COPD patients often have lower airway bacterial colonisation which may be an important stimulus to airway inflammation and thereby modulate exacerbation frequency.

METHODS

Twenty nine patients with COPD (21 men, 16 current smokers) of mean (SD) age 65.9 (7.84) years, forced expiratory volume in 1 second (FEV(1)) 1.06 (0.41) l, FEV(1) % predicted 38.7 (15.2)%, FEV(1)/FVC 43.7 (14.1)%, inhaled steroid dosage 1.<em>20</em> (0.66) mg/day completed daily diary cards for symptoms and peak flow over 18 months. Exacerbation frequency rates were determined from diary card data. Induced sputum was obtained from patients in the stable state, quantitative bacterial culture was performed, and cytokine levels were measured.

RESULTS

Fifteen of the 29 patients (51.7%) were colonised by a possible pathogen: Haemophilus influenzae (53.3%), Streptococcus pneumoniae (33.3%), Haemophilus parainfluenzae (<em>20</em>%), Branhamella catarrhalis (<em>20</em>%), Pseudomonas aeruginosa (<em>20</em>%). The presence of lower airway bacterial colonisation in the stable state was related to exacerbation frequency (p=0.023). Patients colonised by H influenzae in the stable state reported more symptoms and increased sputum purulence at exacerbation than those not colonised. The median (IQR) symptom count at exacerbation in those colonised by H influenzae was 2.00 (2.00-2.65) compared with 2.00 (1.00-2.00) in those not colonised (p=0.03). The occurrence of increased sputum purulence at exacerbation per patient was 0.92 (0.56-1.00) in those colonised with H influenzae and 0.33 (0.00-0.60) in those not colonised (p=0.02). Sputum interleukin (<em>IL</em>)-8 levels correlated with the total bacterial count (rho=0.459, p=0.02).

CONCLUSIONS

Lower airway bacterial colonisation in the stable state modulates the character and frequency of COPD exacerbations.

We investigated whether the proinflammatory T cell cytokines <em>IL</em>-17 and <em>IL</em>-22 are induced by human mycobacterial infection. Remarkably,>><em>20</em>% of specific cytokine-producing CD4(+) T cells in peripheral blood of healthy, mycobacteria-exposed adults expressed <em>IL</em>-17 or <em>IL</em>-22. Specific <em>IL</em>-17- and <em>IL</em>-22-producing CD4(+) T cells were distinct from each other and from Th1 cytokine-producing cells. These cells had phenotypic characteristics of long-lived central memory cells. In patients with tuberculosis disease, peripheral blood frequencies of these cells were reduced, whereas bronchoalveolar lavage fluid contained higher levels of <em>IL</em>-22 protein compared with healthy controls. <em>IL</em>-17 was not detected in this fluid, which may be due to suppression by Th1 cytokines, as PBMC <em>IL</em>-17 production was inhibited by IFN-gamma in vitro. However, Th1 cytokines had no effect on <em>IL</em>-22 production in vitro. Our results imply that the magnitude and complexity of the anti-mycobacterial immune response have historically been underestimated. <em>IL</em>-17- and <em>IL</em>-22-producing CD4(+) T cells may play important roles in the human immune response to mycobacteria.

OBJECTIVE

We set out to test the hypothesis that irritable bowel syndrome (IBS) is characterized by an augmented cellular immune response with enhanced production of proinflammatory cytokines. We further aimed to explore whether symptoms and psychiatric comorbidity in IBS are linked to the release of proinflammatory cytokines.

METHODS

We characterized basal and Escherichia coli lipopolysaccharide (LPS)-induced cytokine production in peripheral blood mononuclear cells (PBMCs) from 55 IBS patients (18 mixed-, 17 constipation-, <em>20</em> diarrhea-predominant) and 36 healthy controls (HCs). PBMCs were isolated by density gradient centrifugation and cultured for 24 hours with or without (1 ng/mL) LPS. Cytokine production (tumor necrosis factor [TNF]-alpha, interleukin [<em>IL</em>]-1beta, and <em>IL</em>-6) was measured by enzyme-linked immunosorbent assay. Abdominal symptoms and psychiatric comorbidities were assessed by using the validated Bowel Disease Questionnaire and the Hospital Anxiety and Depression Scale.

RESULTS

IBS patients showed significantly (P < .017) higher baseline TNF-alpha, IL-1beta, IL-6, and LPS-induced IL-6 levels compared with HCs. Analyzing IBS subgroups, all cytokine levels were significantly (P < .05) higher in diarrhea-predominant IBS (D-IBS) patients, whereas constipation-predominant IBS patients showed increased LPS-induced IL-1beta levels compared with HCs. Baseline TNF-alpha and LPS-induced TNF-alpha and IL-6 levels were significantly higher in patients reporting more than 3 bowel movements per day, urgency, watery stools, and pain associated with diarrhea compared with patients without these symptoms (all P < .05). LPS-induced TNF-alpha production was associated significantly (r = 0.59, P < .001) with anxiety in patients with IBS.

CONCLUSIONS

Patients with D-IBS display enhanced proinflammatory cytokine release, and this may be associated with symptoms and anxiety.

Elevated spontaneous IgG production is characteristic of SLE. To identify the factors that support it, <em>IL</em>-6, a cytokine with an important role in the differentiation of IgG-secreting cells, was studied in SLE patients. Higher than normal levels of <em>IL</em>-6 were found, by a B9 assay, in sera of 63 of 70 patients (p less than 0.05). <em>IL</em>-6 was detected in 36 of 37 active SLE sera in higher titers (p = 0.009) than those for inactive SLE (n = 33), which were higher (p less than 0.05) than healthy controls (n = 15). <em>IL</em>-6 mRNA was detected in freshly isolated PBMC of 11 of 11 patients but not in normal PBMC, whereas <em>IL</em>-1 mRNA was detected only in patients with active disease. <em>IL</em>-6 activity was recovered from PBMC of four SLE patients, but not from four normal donors. By immunoperoxidase, <em>IL</em>-6 was detected in the cytoplasm of SLE monocytes and lymphocytes. When SLE PBMC were grown in short term cultures with no deliberate stimulation, expression of the <em>IL</em>-6 gene declined rapidly. Accordingly, the spontaneous production of IgG by SLE PBMC could be enhanced by exogenous <em>IL</em>-6. Spontaneous IgG production was diminished by <em>20</em> to 65% in the presence of neutralizing antibodies to <em>IL</em>-6, TNF-alpha, or <em>IL</em>-1. In contrast, neutralization of endogenous <em>IL</em>-4 increased production by approximately 40%. Anti-TNF-alpha treatment decreased <em>IL</em>-6 content of PBMC cultures, whereas anti-<em>IL</em>-4 augmented it, and exogenous <em>IL</em>-6 reversed anti-TNF-alpha effects on IgG production. Therefore, it is possible that the neutralization of TNF-alpha and <em>IL</em>-4 affected IgG production by modulating the synthesis/activity of <em>IL</em>-6. These results support the concept that SLE B cell hyperactivity is promoted by dysregulation of endogenous cytokines and suggest that <em>IL</em>-6, in particular, has an important pathogenic role.

The role of eosinophils as effector cells in asthma pathogenesis has been questioned since an anti-interleukin (<em>IL</em>)-5 monoclonal antibody (mepolizumab), which depleted blood and sputum eosinophils, failed to inhibit allergen-induced bronchoconstriction and airway hyperresponsiveness. However, the effect of <em>IL</em>-5 blockade on tissue eosinophils was not examined. We sought to determine whether mepolizumab depletes airway tissue eosinophils and their products. Twenty-four patients with mild asthma received three intravenous doses of either 750 mg of mepolizumab or placebo in a randomized, double-blind, parallel-group fashion over <em>20</em> weeks. Mepolizumab produced a median decrease from baseline of 55% for airway eosinophils (interquartile range, 29-89%; p = 0.009 versus placebo), 52% for bone marrow eosinophils (45-76%, p = 0.003), and 100% for blood eosinophils (range, 67-100%, p = 0.02). Mepolizumab had no appreciable effect on bronchial mucosal staining of eosinophil major basic protein. There were no significant changes in clinical measures of asthma (airway hyperresponsiveness, FEV1, and peak flow recordings) between the mepolizumab and placebo-treated groups. Anti-<em>IL</em>-5 treatment reduces but does not deplete airway or bone marrow eosinophils. The role of the eosinophil remains uncertain. Further clinical studies in asthma with more effective antieosinophil strategies are required.

About 50 human chemokines and nearly <em>20</em> receptors have been identified and characterized in little more than a decade since the discovery of interleukin 8 (<em>IL</em>-8), the first chemotactic cytokine. Research in this field has dramatically changed our understanding of leucocyte traffic in inflammation and immunity. This paper has been written for scientists and practitioners in the field of medicine. It reviews in concise and intelligible form information that I consider useful for understanding the role of chemokines in human pathophysiology. The main areas covered are: (i) the basics of chemokine structures, mode of action, activities and selectivity; (ii) newer aspects of the broad involvement of chemokines in the regulation of immune defence and the housekeeping of the immune system; (iii) the role of chemokines in pathology as illustrated by animal models and studies of human diseases; and (iv) novel therapeutic approaches for a variety of inflammatory conditions, which are based on modulation of chemokine activity.

Formation of the atherosclerotic intima must involve altered metabolism of the elastin-rich arterial extracellular matrix. Proteases potentially involved in these processes remain unclear. This study examined the expression of the potent elastases cathepsins S and K in human atheroma. Normal arteries contained little or no cathepsin K or S. In contrast, macrophages in atheroma contained abundant immunoreactive cathepsins K and S. Intimal smooth muscle cells (SMC), especially cells appearing to traverse the internal elastic laminae, also contained these enzymes. Extracts of atheromatous tissues had approximately twofold greater elastase-specific activity than extracts of uninvolved arteries, mostly due to cysteine proteases. Cultured human SMC displayed no immunoreactive cathepsins K and S and exhibited little or no elastolytic activity when incubated with insoluble elastin. SMC stimulated with the atheroma-associated cytokines <em>IL</em>-1beta or IFN-gamma secreted active cathepsin S and degraded substantial insoluble elastin (15-<em>20</em> microg/10(6) cells/24 h). A selective inhibitor of cathepsin S blocked>> 80% of this elastolytic activity. The presence of cathepsins K and S at sites of vascular matrix remodeling and the ability of SMC and macrophages to use these enzymes to degrade elastin supports a role for elastolytic cathepsins in vessel wall remodeling and identifies novel therapeutic targets in regulating plaque stability.

BACKGROUND

To assess the genetic influence on cytokine production and its contribution to fatal outcome, we determined the capacity to produce tumour necrosis factor-alpha (TNF alpha) and interleukin-10 (IL-10) in families of patients who had had meningococcal disease.

METHODS

We studied 190 first-degree relatives of 61 patients with meningococcal disease; we also studied 26 monozygotic twins. Production of cytokines was determined during endotoxin stimulation of whole-blood samples ex-vivo. Heritability was estimated in a pedigree-based maximum-likelihood model. DNA was typed for the G to A transition polymorphisms at position -308 and -238 in the TNF gene promoter.

RESULTS

Heritability in monozygotic twins was 0.60 for the production of TNF and 0.75 for the production of IL-10. Families with low TNF production had a tenfold increased risk for fatal outcome (OR 8.9, 95% CI 1.8-45), whereas high IL-10 production increased the risk 20-fold (19.5, 2.3-165). Families with both characteristics had the greatest risk. The transition polymorphisms in the TNF gene promoter were not associated with outcome.

CONCLUSIONS

Genetic factors substantially influence production of cytokines. An innate anti-inflammatory cytokine profile may contribute to fatal meningococcal disease.

Herniated intervertebral disc tissue has been shown to produce a number of proinflammatory mediators and cytokines, but there have been no similar studies using discs from patients with discogenic low back pain. We have compared the levels of production of interleukin-6 (<em>IL</em>-6), interleukin-8 (<em>IL</em>-8) and prostaglandin E2 (PGE2) in disc tissue from patients undergoing discectomy for sciatica (63) with that from patients undergoing fusion for discogenic low back pain (<em>20</em>) using an enzyme-linked immunoabsorbent assay. There was a statistically significant difference between levels of production of <em>IL</em>-6 and <em>IL</em>-8 in the sciatica and low back pain groups (p < 0.006 and p < 0.003, respectively). The high levels of proinflammatory mediator found in disc tissue from patients undergoing fusion suggest that production of proinflammatory mediators within the nucleus pulposus may be a major factor in the genesis of a painful lumbar disc.

Chronic antral gastritis following Helicobacter pylori (Hp) infection is characterized by a cellular inflammatory infiltrate whose cytokines may represent a host-dependent factor influencing the outcome of the infection. The pattern of cytokines produced by the immunologically active cells in the gastric antrum was analyzed at the mRNA level in antral biopsies from five Hp-infected patients with duodenal ulcer and three Hp-negative dyspeptic controls. T cell clones were generated from parallel antral biopsies of the same Hp-infected patients and assessed for reactivity to Hp Ags, cytokine profile, and effector functions. Antral biopsies from all Hp-infected patients showed IFN-gamma, TNF-alpha, and <em>IL</em>-12, but not <em>IL</em>-4, mRNA expression, whereas no cytokine mRNA signal was found in the mucosa of controls. A total of 24 out of the 163 CD4+ T cell clones (15%) derived from Hp-infected patients proliferated in response to a Hp lysate; 11 clones (46%) also reacted with Cag-A, 2 with Vac-A, and 1 with urease. Upon Ag stimulation, <em>20</em> out of the 24 Hp-reactive clones (83%) produced IFN-gamma, but not <em>IL</em>-4 or <em>IL</em>-5 (Th1-like), whereas 4 produced IFN-gamma, <em>IL</em>-4, and <em>IL</em>-5 (Th0-like). All Hp-specific clones secreted high levels of TNF-alpha. At low T:B cell ratio, Hp-specific clones expressed Ag-dependent helper function for B cell proliferation and Ig production, whereas at higher T:B cell ratios, 15 Th1 and 2 Th0 clones lysed Ag-pulsed autologous EBV-transformed B cells. Results provide evidence for Hp-specific Th1 effectors in the gastric antrum of Hp-infected patients, where they may play a role in the genesis of either peptic ulcer or Hp-associated gastric B cell lymphoma.

Interleukin-6 (<em>IL</em>-6) is one of several pro-inflammatory cytokines implicated in insulin resistance during infection, cachexia, and obesity. We recently demonstrated that <em>IL</em>-6 inhibits insulin signaling in hepatocytes (Senn, J. J., Klover, P. J., Nowak, I. A., and Mooney, R. A. (<em>20</em>02) Diabetes 51, 3391-3399). Members of the suppressors of cytokine signaling (SOCS) family associate with the insulin receptor (IR), and their ectopic expression inhibits IR signaling. Since several SOCS proteins are induced by <em>IL</em>-6, a working hypothesis is that <em>IL</em>-6-dependent insulin resistance is mediated, at least in part, by induction of SOCS protein(s) in insulin target cells. To examine the involvement of SOCS protein(s) in <em>IL</em>-6-dependent inhibition of insulin receptor signaling, HepG2 cells were treated with <em>IL</em>-6 (<em>20</em> ng/ml) for periods from 1 min to 8 h. <em>IL</em>-6 induced SOCS-3 transcript at 30 min with a maximum effect at 1 h. SOCS-3 protein levels were also markedly elevated at 1 h. Transcript and protein levels returned to near basal levels by 2 h. SOCS-3 induction by <em>IL</em>-6 paralleled <em>IL</em>-6-dependent inhibition of IR signal transduction. Ectopically expressed SOCS-3 associated with the IR and suppressed insulin-dependent receptor autophosphorylation, insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase, and activation of Akt. SOCS-3 was also a direct inhibitor of insulin receptor autophosphorylation in vitro. In mice exposed to <em>IL</em>-6 for 60-90 min, hepatic SOCS-3 expression was increased. This was associated with inhibition of hepatic insulin-dependent receptor autophosphorylation and IRS-1 tyrosine phosphorylation. These data suggest that induction of SOCS-3 in liver may be an important mechanism of <em>IL</em>-6-mediated insulin resistance.

Virtually of the all recent therapeutic interventions for treating sepsis have failed to improve survival. One potential explanation is that the heterogeneity of the immune response to the septic challenge is such that only a portion of the patients die as a result of excessive inflammation. The clinical trials lacked power because traditional measurements do not accurately identify these patients. Previous work has shown that higher levels of interleukin (<em>IL</em>)-6 are found in those mice that die from septic peritonitis; therefore, we sought to determine whether <em>IL</em>-6 measured 6 h after surgery could predict outcome. Adult, female BALB/c mice (n = 79) were subjected to cecal ligation and puncture with a 21-gauge needle and treated with imipenem in D5W every 12 h for 5 days, resulting in a homogenous population at the outset. Six hours after surgery, <em>20</em> microL of blood was obtained from the tail vein to measure <em>IL</em>-6. Mortality was followed for 21 days. Overall 3-day survival was 77%, and 21-day mortality was 56%. Plasma <em>IL</em>-6 levels >2,000 pg/mL were determined to predict mortality within the first 3 days with a sensitivity of 58% and specificity of 97%. To further refine the mortality prediction, body weight and a complete blood count were performed 24 hours after cecal ligation and puncture. Discriminate analysis indicated that a weighted formula combining body mass, lymphocyte, and platelet count would predict death with sensitivity of 83% and a specificity of 79%. We tested the value of the <em>IL</em>-6 prediction by surgically resecting the cecum in those animals with <em>IL</em>-6>> <em>20</em>00 pg/mL, which resulted in a significant improvement in survival. These data demonstrate that <em>IL</em>-6 measured 6 h after injury accurately predicts mortality resulting from experimental sepsis. This measurement may be determined quickly so that therapy may be targeted only to those individuals at significant risk of dying and initiated within sufficient time to be effective.

Stress conditions and proinflammatory cytokines activate the c-Jun NH2-terminal kinase (JNK), a member of the stress-activated group of mitogen-activated protein kinases (MAPKs). We recently demonstrated that inhibition of JNK signaling with the use of the islet-brain (IB) 1 and 2 proteins prevented interleukin (<em>IL</em>)-1beta-induced pancreatic beta-cell death. Bioactive cell-permeable peptide inhibitors of JNK were engineered by linking the minimal <em>20</em>-amino acid inhibitory domains of the IB proteins to the 10-amino acid HIV-TAT sequence that rapidly translocates inside cells. Kinase assays indicate that the inhibitors block activation of the transcription factor c-Jun by JNK. Addition of the peptides to the insulin-secreting betaTC-3 cell line results in a marked inhibition of <em>IL</em>-1beta-induced c-jun and c-fos expression. The peptides protect betaTC-3 cells against apoptosis induced by <em>IL</em>-1beta. All-D retro-inverso peptides penetrate cells as efficiently as the L-enantiomers, decrease c-Jun activation by JNK, and remain highly stable inside cells. These latter peptides confer full protection against <em>IL</em>-1beta-induced apoptosis for up to 2 weeks of continual treatment with <em>IL</em>-1beta. These data establish these bioactive cell-permeable peptides as potent pharmacological compounds that decrease intracellular JNK signaling and confer long-term protection to pancreatic beta-cells from <em>IL</em>-1beta-induced apoptosis.

We have investigated the level of TCR occupancy required to elicit different biological responses in human CTL clones specific for an influenza matrix peptide. Specific cytotoxicity could be detected at extremely low peptide concentrations (10(-12) to 10(-15) M). However, IFN-gamma production, responsiveness to <em>IL</em>-2 and Ca++ fluxes were observed only at peptide concentrations>> 10(-9) M, while autonomous proliferation required even higher peptide concentrations. In parallel experiments we measured TCR downregulation to estimate the number of TCRs triggered. We observed that at low peptide concentrations, where only cytotoxicity is triggered, TCR downregulation was hardly detectable. Conversely, induction of IFN-gamma production and proliferation required triggering of at least <em>20</em>-50% of TCRs. Taken together these results indicate that a single CTL can graduate different biological responses as a function of antigen concentration and that killing of the specific target does not necessarily result in full activation.

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of autologous T cells engineered to express an affinity-enhanced T cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4 × 10(9) engineered T cells 2 d after autologous stem cell transplant. Infusions were well tolerated without clinically apparent cytokine-release syndrome, despite high <em>IL</em>-6 levels. Engineered T cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, in accordance with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of <em>20</em> patients (80%) with advanced disease, with a median progression-free survival of 19.1 months. NY-ESO-1-LAGE-1 TCR-engineered T cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.

To determine whether cytokines are generated in vivo in subjects with asthma, we have measured cytokine levels (tumor necrosis factor [TNF], granulocyte-macrophage-colony-stimulating factor [GM-CSF], interleukin [<em>IL</em>]-1 alpha, <em>IL</em>-1 beta, <em>IL</em>-2, <em>IL</em>-4, and <em>IL</em>-6) in the airways of subjects with symptomatic (N = 24) and asymptomatic (N = 9) asthma with immunoassays (GM-CSF, <em>IL</em>-1 alpha, <em>IL</em>-1 beta, <em>IL</em>-2, and <em>IL</em>-4) or bioassays (TNF and <em>IL</em>-6) and the polymerase chain reaction (<em>IL</em>-1 beta and TNF). Significant levels of TNF (578 +/- 917 pg/ml versus 24 +/- 29 pg/ml) (p = 0.01), GM-CSF (24 +/- 41 pg/ml versus less than 8 pg/ml) (p = 0.02), and <em>IL</em>-6 (225 +/- 327 pg/ml versus 7 +/- 12 pg/ml) (p = 0.01), but not <em>IL</em>-1 alpha or <em>IL</em>-4, were detected in the bronchoalveolar lavage fluid (BALF) of patients with symptomatic compared with BALF of patients with asymptomatic asthma. Levels of <em>IL</em>-1 beta (266 +/- 270 pg/ml versus less than <em>20</em> pg/ml) (p = 0.001) and <em>IL</em>-2 (1.4 +/- 2.8 ng/ml versus less than 0.3 ng/ml) (p = 0.05) in BALF in patients with symptomatic compared with that in BALF levels in patients with asymptomatic asthma suggested activation of alveolar macrophages and T cells. Thus, in episodes of asthma, several cytokines, including TNF, GM-CSF, <em>IL</em>-1 beta, <em>IL</em>-2, and <em>IL</em>-6 are detectable in BALF.

Understanding the mechanisms of immune cell migration to multiple sclerosis lesions offers significant therapeutic potential. This study focused on the chemokines CXCL12 (SDF-1) and CXCL13 (BCA-1), both of which regulate B cell migration in lymphoid tissues. We report that immunohistologically CXCL12 was constitutively expressed in CNS parenchyma on blood vessel walls. In both active and chronic inactive multiple sclerosis lesions CXCL12 protein was elevated and detected on astrocytes and blood vessels. Quantitative PCR demonstrated that CXCL13 was produced in actively demyelinating multiple sclerosis lesions, but not in chronic inactive lesions or in the CNS of subjects who had no neurological disease. CXCL13 protein was localized in perivascular infiltrates and scattered infiltrating cells in lesion parenchyma. In the CSF of relapsing-remitting multiple sclerosis patients, both CXCL12 and CXCL13 were elevated. CXCL13, but not CXCL12, levels correlated strongly with intrathecal immunoglobulin production as well as the presence of B cells, plasma blasts and T cells. About <em>20</em>% of CSF CD4+ cells and almost all B cells expressed the CXCL13 receptor CXCR5. In vitro, CXCL13 was produced by monocytes and at much higher levels by macrophages. CXCL13 mRNA and protein expression was induced by TNFalpha and <em>IL</em>-1beta but inhibited by <em>IL</em>-4 and IFNgamma. Together, CXCL12 and CXCL13 are elevated in active multiple sclerosis lesions and CXCL12 also in inactive lesions. The consequences of CXCL12 up-regulation could be manifold. CXCL12 localization on blood vessels indicates a possible role in leucocyte extravasation, and CXCL12 may contribute to plasma cell persistence since its receptor CXCR4 is retained during plasma cell differentiation. CXCL12 may contribute to axonal damage as it can become a neurotoxic mediator of cleavage by metalloproteases, which are present in multiple sclerosis lesions. The strong linkage of CXCL13 to immune cells and immunoglobulin levels in CSF suggests that this is one of the factors that attract and maintain B and T cells in inflamed CNS lesions. Therefore, both CXCL13 and CXCR5 may be promising therapeutic targets in multiple sclerosis.

Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10-<em>20</em>-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)gamma production and an enhanced secondary response to antigenic challenge. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNgamma, and <em>IL</em>-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity.

We show here that <em>IL</em>-17-secreting CD4 T (Th)17 and CD8 T (Tc)17 effector cells are found in the lung following primary challenge with influenza A and that blocking Ab to <em>IL</em>-17 increases weight loss and reduces survival. Tc17 effectors can be generated in vitro using naive CD8 T cells from OT-I TCR-transgenic mice. T cell numbers expand <em>20</em>-fold and a majority secretes <em>IL</em>-17, but little IFN-gamma. Many of the <em>IL</em>-17-secreting cells also secrete TNF and some secrete <em>IL</em>-2. Tc17 are negative for granzyme B, perforin message, and cytolytic activity, in contrast to Tc1 effectors. Tc17 populations express message for orphan nuclear receptor gammat and FoxP3, but are negative for T-bet and GATA-3 transcription factors. The FoxP3-positive, <em>IL</em>-17-secreting and IFN-gamma-secreting cells represent three separate populations. The IFN-gamma-, granzyme B-, FoxP3-positive cells and cells positive for <em>IL</em>-22 come mainly from memory cells and decrease in number when generated from CD44(low) rather than unselected CD8 T cells. Cells of this unique subset of CD8 effector T cells expand greatly after transfer to naive recipients following challenge and can protect them against lethal influenza infection. Tc17 protection is accompanied by greater neutrophil influx into the lung than in Tc1-injected mice, and the protection afforded by Tc17 effectors is less perforin but more IFN-gamma dependent, implying that different mechanisms are involved.

<em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, <em>IL</em>-24, and <em>IL</em>-26 are members of the <em>IL</em>-10 family of cytokines that have been shown to be up-regulated in psoriatic skin. Contrary to <em>IL</em>-10, these cytokines signal using receptor complex R1 subunits that are preferentially expressed on cells of epithelial origin; thus, we henceforth refer to them as the <em>IL</em>-<em>20</em> subfamily cytokines. In this study, we show that primary human keratinocytes (KCs) express receptors for these cytokines and that <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, and <em>IL</em>-24 induce acanthosis in reconstituted human epidermis (RHE) in a dose-dependent manner. These cytokines also induce expression of the psoriasis-associated protein S100A7 and keratin 16 in RHE and cause persistent activation of Stat3 with nuclear localization. <em>IL</em>-22 had the most pronounced effects on KC proliferation and on the differentiation of KCs in RHE, inducing a decrease in the granular cell layer (hypogranulosis). Furthermore, gene expression analysis performed on cultured RHE treated with these cytokines showed that <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, and <em>IL</em>-24 regulate many of these same genes to variable degrees, inducing a gene expression profile consistent with inflammatory responses, wound healing re-epithelialization, and altered differentiation. Many of these genes have also been found to be up-regulated in psoriatic skin, including several chemokines, beta-defensins, S100 family proteins, and kallikreins. These results confirm that <em>IL</em>-<em>20</em> subfamily cytokines are important regulators of epidermal KC biology with potentially pivotal roles in the immunopathology of psoriasis.