BACKGROUND

Cystic fibrosis (CF) is characterized by bronchoalveolar neutrophilia and submucosal lymphocytosis. We hypothesized that Th17 lymphocytes are part of this submucosal infiltrate.

OBJECTIVE

Quantification and phenotyping of the lymphocytic infiltrate in the bronchial submucosa of patients with CF (n = 53, of which <em>20</em> were newly diagnosed), non-CF bronchiectasis (n = 17), and healthy control subjects (n = 13).

METHODS

We measured IL-17 levels in bronchoalveolar lavage and CD4(+), CD8(+), and IL-17(+) cell counts in endobronchial biopsies. Correlations were made with infection status and other inflammatory markers. Potential cellular sources of IL-17 were determined by double staining.

RESULTS

IL-17(+) cell counts (median [interquartile range] cells/mm(2)) were significantly higher in patients with established CF (<em>20</em>5 [115-551]) and non-CF bronchiectasis (245 [183-436]) than in control subjects (53 [12-82]) (P < 0.01 for both). Patients with newly diagnosed CF had intermediate counts (171 [91-252]). IL-17-positive CD4(+) T cells, γδT cells, natural killer T cells, and neutrophils were identified. Bronchoalveolar lavage IL-17 levels (pg/ml) were highest in established CF (14.6 [2.2-38.4]), low in newly diagnosed CF and control subjects (1.7 [1.7-1.74]; 1.7 [1.7-3]), and intermediate in non-CF bronchiectasis (9.1 [1.7-34] pg/ml) (Kruskal-Wallis P = 0.001). There was a significant correlation between IL-17 and neutrophil counts (P < 0.001, R = 0.6) as well as IL-4 (P < 0.001, R = 0.84).

CONCLUSIONS

Th17 lymphocytes are present in the airway submucosa in CF, even in a young, newly diagnosed group. Other IL-17(+) cells include neutrophils, γδ T cells, and natural killer T cells.

cDNAs encoding TCR alpha- and beta-chains specific for HLA-A2-restricted cancer-testis Ag NY-ESO-1 were cloned using a 5'RACE method from RNA isolated from a CTL generated by in vitro stimulation of PBMC with modified NY-ESO-1-specific peptide (p157-165, 9V). Functionality of the cloned TCR was confirmed by RNA electroporation of primary PBL. cDNA for these alpha- and beta-chains were used to construct a murine stem cell virus-based retroviral vector, and high titer packaging cell lines were generated. Gene transfer efficiency in primary T lymphocytes of up to 60% was obtained without selection using a method of precoating retroviral vectors onto culture plates. Both CD4(+) and CD8(+) T cells could be transduced at the same efficiency. High avidity Ag recognition was demonstrated by coculture of transduced lymphocytes with target cells pulsed with low levels of peptide ((<em>20</em> pM). TCR-transduced CD4 T cells, when cocultured with NY-ESO-1 peptide pulsed T2 cells, could produce IFN-gamma, GM-CSF, <em>IL</em>-4, and <em>IL</em>-10, suggesting CD8-independent, HLA-A2-restricted TCR activation. The transduced lymphocytes could efficiently recognize and kill HLA-A2- and NY-ESO-1-positive melanoma cell lines in a 4-h (51)Cr release assay. Finally, transduced T cells could efficiently recognize NY-ESO-1-positive nonmelanoma tumor cell lines. These results strongly support the idea that redirection of normal T cell specificity by TCR gene transfer can have potential applications in tumor adoptive immunotherapy.

OBJECTIVE

To investigate the in vivo effect of recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) on the development of lesions and the expression of metalloproteases in the canine experimental osteoarthritis (OA) model.

METHODS

The right anterior cruciate ligament was sectioned percutaneously in 3 groups of dogs. The control group (n = 5) received an intraarticular injection of sterile physiologic saline (1 ml) twice weekly for 4 weeks starting on the day of surgery. The remaining 2 groups received intraarticular injections of either 2 mg (n = 6) or 4 mg (n = 5) rHuIL-1Ra in 1 ml of physiologic saline according to the same schedule as the first group. All dogs were killed 4 weeks after surgery. The macroscopic appearance of femoral condyle osteophytes and the size and severity of cartilage lesions on femoral condyles and tibial plateaus were evaluated, as were the histologic features of cartilage and synovial membrane. Levels of collagenase-1 and stromelysin-1 messenger RNA expression in cartilage and synovium were determined by Northern blotting.

RESULTS

Recombinant human <em>IL</em>-1Ra exerted a dose-dependent protective effect on the development of osteophytes and cartilage lesions in vivo. Treatment with rHu<em>IL</em>-1Ra reduced the incidence (saline-treated group 70%, 2 mg rHu<em>IL</em>-1Ra-treated group 42%, 4 mg rHu<em>IL</em>-1Ra-treated group <em>20</em>%) and size (saline-treated group 2.3 +/- 0.7 mm [mean +/- SEM], 2 mg rHu<em>IL</em>-1Ra-treated group 0.7 +/- 0.3 mm, 4 mg rHu<em>IL</em>-1Ra-treated group 0.5 +/- 0.3 mm) of femoral condyle osteophytes. In addition, a dose-dependent decrease in the size (saline-treated group 24.40 +/- 8.17 mm2, 2 mg rHu<em>IL</em>-1Ra-treated group <em>20</em>.90 +/- 8.01 mm2, 4 mg rHu<em>IL</em>-1Ra-treated group 7.70 +/- 5.16 mm2) and the grade (0-4 scale; saline-treated group 1.<em>20</em> +/- 0.29, 2 mg rHu<em>IL</em>-1Ra-treated group 1.00 +/- 0.26, 4 mg rHu<em>IL</em>-1Ra-treated group 0.30 +/- 0.21) of the tibial plateau cartilage lesions was found, with a significant difference (P < 0.04) reached only with 4 mg rHu<em>IL</em>-1Ra. Similarly, the histologic lesions in dogs treated with 4 mg rHu<em>IL</em>-1Ra (Mankin scale; mean +/- SEM 2.95 +/- 0.53) were significantly less severe (P < 0.002) compared with those in the saline-treated group (4.95 +/- 0.54). Importantly, rHu<em>IL</em>-1Ra treatment led to a significant reduction (P < 0.005) of collagenase-1 expression in OA cartilage.

CONCLUSIONS

This study demonstrated that intraarticular injections of rHuIL-1Ra can protect against the development of experimentally induced OA lesions. This effect could result, at least in part, from a reduction of collagenase-1 expression. However, other catabolic processes involved in the degradation of OA cartilage may also be affected.

Clinical studies using biological response modifiers in cancer therapy have shown that the major dose-limiting toxic effects are hypotension and diffuse microvascular leakage. The cause and pathophysiology of this hypotension remains unknown. Previous experiments have demonstrated that a number of cell types, including endothelial cells, neutrophils, and macrophages, can secrete a potent hypotensive agent--endothelium-derived relaxing factor, which has recently been identified as nitric oxide. In this study, we tested interferon gamma, tumor necrosis factor, interleukin-1, interleukin-2, muramyl dipeptide, and endotoxin for their effects on production of nitrogen oxides by endothelial cells. Interferon gamma, in combination with tumor necrosis factor, interleukin-1 (<em>IL</em>-1), or endotoxin, induced murine brain endothelial cells to secrete nitrites (<em>20</em>-45 microM within 48 hr), which are breakdown products of nitric oxide. Nitrite production was blocked by incubation of endothelial cells in medium without L-arginine, a substrate for nitric-oxide synthase. Accumulation of nitrites was also inhibited by addition of NG-monomethyl-L-arginine (L-NMMA), which acts as a competitive inhibitor of this enzyme. The inhibitory effects of L-NMMA were reversed by addition of excess L-arginine. These results suggest (a) that endothelial cells produce nitric oxide in response to immunomodulators and (b) that endothelial cell-derived nitric oxide plays a role in the development of hypotension in patients treated with tumor necrosis factor or interleukins. Furthermore, administration of substrate analogues such as L-NMMA may favorably alter the toxicity associated with these immunomodulators and result in a higher maximum tolerated dose, with subsequent improvement in the antitumor activity.

Th1 and Th17 cells are characterized by their expression of IFN-gamma or <em>IL</em>-17, respectively. The finding of Th cells producing both <em>IL</em>-17 and IFN-gamma suggested, however, that certain Th cells may modify their selective cytokine expression. In this study, we examined changes in cytokine expression in an experimental system in which polarized Th1 or Th17 cells specific against hen egg lysozyme induce ocular inflammation in recipient mice expressing hen egg lysozyme in their eyes. Whereas only IFN-gamma was expressed in eyes of Th1 recipient mice, substantial proportions of donor cells expressed IFN-gamma or both IFN-gamma and <em>IL</em>-17 in Th17 recipient eyes. The possibility that nonpolarized cells in Th17 preparations were responsible for expression of IFN-gamma or IFN-gamma/<em>IL</em>-17 in Th17 recipient eyes was contradicted by the finding that the proportions of such cells were larger in recipients of Th17 preparations with <em>20</em>-25% nonpolarized cells than in recipients of 35-40% preparations. Moreover, whereas incubation in vitro of Th1 cells with Th17-polarizing mixture had no effect on their phenotype, incubation of Th17 with Th1-polarizing mixture, or in the absence of cytokines, converted most of these cells into IFN-gamma or IFN-gamma/<em>IL</em>-17-expressing cells. In addition, Th17 incubated with the Th1 mixture expressed T-bet, whereas no ROR-gamma t was detected in Th1 incubated with Th17 mixture. Thus, polarized Th1 cells retain their phenotype in the tested systems, whereas Th17 may switch to express IFN-gamma or IFN-gamma/<em>IL</em>-17 following activation in the absence of cytokines, or exposure to certain cytokine milieus at the inflammation site or in culture.

BACKGROUND

Proinflammatory cytokines have been reported to be elevated in individuals experiencing chronic stress as well as in those with major depressive disorder. Much less is known about cytokines in anxiety disorders such as posttraumatic stress disorder (PTSD) and panic disorder (PD). We hypothesized that PD and PTSD would be associated with a generalized proinflammatory cytokine signature.

METHODS

We utilized Luminex technology to examine <em>20</em> cytokines and chemokines in serum from 48 well-characterized individuals with a primary DSM-IV PD or PTSD diagnosis, and 48 age- and gender-matched healthy controls. We conservatively employed a Bonferroni correction for multiple testing (alpha=.05/<em>20</em>=.0025).

RESULTS

Individuals with primary PTSD or PD had significantly elevated median peripheral cytokine levels for 18 of <em>20</em> different cytokines compared to age- and gender-matched healthy controls (all P<.0025). To assess for the presence of a generalized proinflammatory state, we also examined the proportion of subjects with detectable levels of at least six of nine common proinflammatory cytokines and chemokines (IL-6, IL-1alpha, IL-1beta, IL-8, MCP-1, MIP-1alpha, Eotaxin, GM-CSF, and IFN-alpha). For men and women, 87% of anxiety patients had six or more detectable levels of these proinflammatory cytokines, compared with only 25% of controls (Fisher's Exact Test (FET) P=.000). Confirmatory analysis of the subset of individuals without current psychiatric medication use or comorbid depression was of comparable significance.

CONCLUSIONS

These findings suggest that a generalized inflammatory state may be present in individuals with PD or PTSD.

Cytokines with bone-resorbing activity include <em>IL</em> 1 beta (pI 7), <em>IL</em> 1 alpha (pI 5), tumor necrosis factor (TNF), and lymphotoxin (LT). Possible interaction between <em>IL</em> 1 beta, the major mediator with osteoclast-activating factor (OAF) activity, and other cytokines was studied. By itself, <em>IL</em> 1 beta was 13-fold more potent than <em>IL</em> 1 alpha and 1000-fold more potent than either TNF or LT in stimulating bone resorption. Suboptimal concentrations of <em>IL</em> 1 beta or <em>IL</em> 1 alpha in combination with suboptimal concentrations of TNF or LT resulted in synergistic bone-resorptive responses (1.5 to 10 times the expected responses if their effects were additive). Synergy between either form of <em>IL</em> 1 and TNF or LT resulted in a twofold increase in activity of <em>IL</em> 1, and a 100-fold increase in activity of TNF or LT. However, even with optimal synergy, <em>IL</em> 1 beta remained <em>20</em>-fold more potent in inducing bone resorption than TNF or LT. Because <em>IL</em> 1 beta is considerably more potent than TNF and LT in stimulating bone resorption either alone or under synergistic conditions, it is unlikely that TNF and LT are responsible for more than a minor proportion of the total bone-resorbing activity formerly referred to as OAF.

BACKGROUND

Air pollution is linked to central nervous system disease, but the mechanisms responsible are poorly understood.

OBJECTIVE

Here, we sought to address the brain-region-specific effects of diesel exhaust (DE) and key cellular mechanisms underlying DE-induced microglia activation, neuroinflammation, and dopaminergic (DA) neurotoxicity.

METHODS

Rats were exposed to DE (2.0, 0.5, and 0 mg/m3) by inhalation over 4 weeks or as a single intratracheal administration of DE particles (DEP; <em>20</em> mg/kg). Primary neuron-glia cultures and the HAPI (highly aggressively proliferating immortalized) microglial cell line were used to explore cellular mechanisms.

RESULTS

Rats exposed to DE by inhalation demonstrated elevated levels of whole-brain IL-6 (interleukin-6) protein, nitrated proteins, and IBA-1 (ionized calcium-binding adaptor molecule 1) protein (microglial marker), indicating generalized neuroinflammation. Analysis by brain region revealed that DE increased TNFα (tumor necrosis factor-α), IL-1β, IL-6, MIP-1α (macrophage inflammatory protein-1α) RAGE (receptor for advanced glycation end products), fractalkine, and the IBA-1 microglial marker in most regions tested, with the midbrain showing the greatest DE response. Intratracheal administration of DEP increased microglial IBA-1 staining in the substantia nigra and elevated both serum and whole-brain TNFα at 6 hr posttreatment. Although DEP alone failed to cause the production of cytokines and chemokines, DEP (5 μg/mL) pretreatment followed by lipopolysaccharide (2.5 ng/mL) in vitro synergistically amplified nitric oxide production, TNFα release, and DA neurotoxicity. Pretreatment with fractalkine (50 pg/mL) in vitro ameliorated DEP (50 μg/mL)-induced microglial hydrogen peroxide production and DA neurotoxicity.

CONCLUSIONS

Together, these findings reveal complex, interacting mechanisms responsible for how air pollution may cause neuroinflammation and DA neurotoxicity.

OBJECTIVE

To compare the singular and combined effects of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-17 on messenger RNA (mRNA) expression, translation, and secretion of IL-6, IL-8, and IL-1beta in fibroblasts.

METHODS

Fibroblasts were stimulated with the relevant cytokine(s), pulse labeled with 35S-methionine, and the newly synthesized proteins were immunoprecipitated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gene expression was determined by Northern blot analysis. Secreted proteins were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTS

IL-17 alone was a weaker stimulator of the transcription, translation, and secretion of other interleukins than was TNFalpha or IL-1beta. IL-17 (10 ng/ml) stimulated the expression of IL-6 mRNA by 1.3-fold, while TNFalpha (1 ng/ml) increased it by 3.7-fold, and IL-1beta (0.1 ng/ml) increased it by >30-fold. Unlike TNFalpha and IL-1beta, IL-17 hardly affected the expression of IL-8 and IL-1beta mRNA. Translation of IL-6 was 6.2 times greater with IL-17, but TNFalpha and IL-1beta stimulated it 28.9- and 174-fold, respectively. ELISA-measured secretion of IL-6 and IL-8 increased by 6.7 and 5.8 times, respectively, with IL-17, compared with 52 and 269 times with TNFalpha stimulation and 1,356 and 1,084 times with IL-1beta stimulation. Yet, when IL-17 was combined with other cytokines, these activities were stimulated much beyond the sum of the individual effects. The combination of IL-17 and TNFalpha induced the expression of IL-6 or IL-1beta mRNA 7 times more than their additive stimulation, and that of IL-8 mRNA 3.8 times more. Likewise, the secretion of IL-6 and IL-8 was 20 times and 5 times higher, respectively, than expected. This synergism started after 4 hours of combined treatment, and decayed after 24-48 hours regardless of cytokine presence. It could be blocked with anti-IL-17 but not with anti-IL-1.

CONCLUSIONS

Our findings suggest that the primary role of IL-17 is to synergize with TNFalpha and to fine-tune the inflammation process. Therefore, IL-17 may be a potential target for therapeutic intervention.

BACKGROUND

The incidence of esophageal adenocarcinoma has increased significantly in the western world over the last <em>20</em> yr. Most cases arise in a background of chronic gastroesophageal reflux, and specialized intestinal metaplasia in Barrett's esophagus is frequently an antecedent phenotype or evident in association with adenocarcinoma. The molecular events that characterize the pathway from inflammation to metaplasia to dysplasia and adenocarcinoma are poorly understood.

OBJECTIVE

To examine the expression of the proinflammatory cytokines IL-8 and IL-1beta along the esophagitis, metaplasia, dysplasia, and adenocarcinoma pathway, and to correlate this with histological changes and expression of the transcription factor NF-kappaB.

METHODS

Fresh biopsy specimens were collected from patients with reflux esophagitis (n=15), Barrett's esophagus (n=35), Barrett's adjacent to adenocarcinoma (n=8), and esophageal adenocarcinoma (n=35). IL-8 and IL-1beta expression were measured using enzyme-linked immunosorbent assay. NF-kappaB expression was measured by electrophoretic mobility shift assay.

RESULTS

Elevated expression of NF-kappaB was found in 2 (13%) out of 15 patients with reflux esophagitis, 21 (60%) out of 35 patients with Barrett's esophagus, and 28 (80%) out of 35 patients with esophageal adenocarcinoma. All 5 patients with Barrett's esophagus and high-grade dysplasia showed elevated expression of NF-kappaB. IL-8 and IL-1beta were significantly increased in esophagitis, Barrett's, and adenocarcinoma compared with squamous epithelium, and in adenocarcinoma compared with all other groups. There was a stepwise increase in the expression of IL-8, IL-1beta, and NF-kappaB from normal through Barrett's epithelium to adenocarcinoma in eight cases of esophageal adenocarcinoma. The levels of both IL-8 and IL-1beta in adenocarcinoma patients correlated with stage of disease. Patients with adenocarcinoma who were NF-kappaB positive had significantly higher levels of both IL-8 (p=0.04) and IL-1beta (p=0.03) compared to adenocarcinoma patients who were NF-kappaB negative.

CONCLUSIONS

The proinflammatory cytokines IL-8 and IL-1beta are elevated in esophagitis and Barrett's epithelium, and markedly elevated in adenocarcinoma. NF-kappaB activation is infrequent in esophagitis, but is increased in Barrett's epithelium and adenocarcinoma. The association of NF-kappaB activation with cytokine upregulation was only evident in patients with adenocarcinoma. These patterns may play an important role in Barrett's inflammation and tumourigenesis, and inhibition of the NF-kappaB/proinflammatory cytokine pathway may be an important target for future chemoprevention strategies.

BACKGROUND

Myeloid derived suppressor cells (MDSC) are important regulators of immune responses. We evaluated the mechanistic role of MDSC depletion on antigen presenting cell (APC), NK, T cell activities and therapeutic vaccination responses in murine models of lung cancer.

RESULTS

Individual antibody mediated depletion of MDSC (anti-Gr1 or anti-Ly6G) enhanced the antitumor activity against lung cancer. In comparison to controls, MDSC depletion enhanced the APC activity and increased the frequency and activity of the NK and T cell effectors in the tumor. Compared to controls, the anti-Gr1 or anti-Ly6G treatment led to increased: (i) CD8 T cells, (ii) NK cells, (iii) CD8 T or NK intracytoplasmic expression of IFNγ, perforin and granzyme (iv) CD3 T cells expressing the activation marker CD107a and CXCR3, (v) reduced CD8 T cell <em>IL</em>-10 production in the tumors (vi) reduced tumor angiogenic (VEGF, CXCL2, CXCL5, and Angiopoietin1&2) but enhanced anti-angiogenic (CXCL9 and CXCL10) expression and (vii) reduced tumor staining of endothelial marker Meca 32. Immunocytochemistry of tumor sections showed reduced Gr1 expressing cells with increased CD3 T cell infiltrates in the anti-Gr1 or anti-Ly6G groups. MDSC depletion led to a marked inhibition in tumor growth, enhanced tumor cell apoptosis and reduced migration of the tumors from the primary site to the lung compared to controls. Therapeutic vaccination responses were enhanced in vivo following MDSC depletion with 50% of treated mice completely eradicating established tumors. Treated mice that rejected their primary tumors acquired immunological memory against a secondary tumor challenge. The remaining 50% of mice in this group had <em>20</em> fold reductions in tumor burden compared to controls.

CONCLUSIONS

Our data demonstrate that targeting MDSC can improve antitumor immune responses suggesting a broad applicability of combined immune based approaches against cancer. This multifaceted approach may prove useful against tumors where MDSC play a role in tumor immune evasion.

Establishing direct and causal relationships among the confederacy of activated cell types present in psoriasis has been hampered by lack of an animal model. Within psoriatic plaques there are hyperplastic keratinocytes, infiltrating immunocytes, and activated endothelial cells. The purpose of this study was to determine if psoriasis is primarily a disorder of keratinocytes or the immune system. Using a newly developed experimental system in which full-thickness human skin is orthotopically transferred onto severe combined immunodeficient mice, autologous immunocytes were injected into dermis, and the resultant phenotype characterized by clinical, histologic, and immunophenotypic analyses. Engraftment of samples included both uninvolved/ symptomless (PN) skin removed from patients with psoriasis elsewhere, or from healthy individuals with no skin disease (NN skin). In 10 different experiments involving 6 different psoriasis patients, every PN skin was converted to a full-fledged psoriatic plaque skin by injection of autologous blood-derived immunocytes. In all but one psoriatic patient, the immunocytes required preactivation with <em>IL</em>-2 and superantigens to convert PN skin into psoriatic plaque skin. In every case, resultant plaques were characterized by visible presence of flaking and thickened skin, loss of the granular cell layer, prominent elongation of rete pegs with a dermal angiogenic tissue reaction, and infiltration within the epidermis by T cells. Lesional skin displayed <em>20</em> different antigenic determinants of the psoriatic phenotype. None of the four NN skin samples injected with autologous immunocytes converted to psoriatic plaques. We conclude that psoriasis is caused primarily by the ability of pathogenetic blood-derived immunocytes to induce secondary activation and disordered growth of endogenous cutaneous cells including keratinocytes and vascular endothelium.

T cell suppressor factor produced by human glioblastoma cells inhibits T cell proliferation in vitro and more specifically interferes with interleukin-2 (<em>IL</em>-2)-dependent T cell growth. Here we report the purification of this factor from conditioned medium of the human glioblastoma cell line 308. Amino-terminal sequence analysis of the 12.5-kd protein demonstrates that eight out of the first <em>20</em> amino acids are identical to human transforming growth factor-beta. Purified glioblastoma-derived T cell suppressor factor and transforming growth factor-beta from porcine platelets inhibit both <em>IL</em>-2-induced proliferation of ovalbumin-specific T helper cells and lectin-induced thymocyte proliferation with similar specific activities. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.

CCL20, like human beta-defensin (hBD)-2, is a potent chemoattractant for CCR6-positive immature dendritic cells and T cells in addition to recently found antimicrobial activities. We previously demonstrated that IL-17 is the most potent cytokine to induce an apical secretion and expression of hBD-2 by human airway epithelial cells, and the induction is JAK/NF-kappaB-dependent. Similar to hBD-2, IL-17 also induced CCL20 expression, but the nature of the induction has not been elucidated. Compared with a panel of cytokines (IL-1alpha, 1beta, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, IFN-gamma, GM-CSF, and TNF-alpha), IL-17 was as potent as IL-1alpha, 1beta, and TNF-alpha, with a time- and dose-dependent phenomenon in stimulating CCL20 expression in both well-differentiated primary human and mouse airway epithelial cell culture systems. The stimulation was largely dependent on the treatment of polarized epithelial cultures from the basolateral side with IL-17, achieving an estimated 4- to 10-fold stimulation at both message and protein levels. More than 90% of induced CCL20 secretion was toward the basolateral compartment (23.02 +/- 1.11 ng/chamber/day/basolateral vs 1.82 +/- 0.82 ng/chamber/day/apical). Actinomycin D experiments revealed that enhanced expression did not occur at mRNA stability. Inhibitor studies showed that enhanced expression was insensitive to inhibitors of JAK/STAT, p38, JNK, and PI3K signaling pathways, but sensitive to inhibitors of MEK1/2 and NF-kappaB activation, suggesting a MEK/NF-kappaB-based mechanism. These results suggest that IL-17 can coordinately up-regulate both hBD-2 and CCL20 expressions in airways through differentially JAK-dependent and -independent activations of NF-kappaB-based transcriptional mechanisms, respectively.

Despite optimal suppression of HIV replication, restoration of CD4(+) T cells is not always achieved in antiretroviral therapy-treated individuals. Defective CD4 recovery in immunologic nonresponders is possibly associated with TLR-mediated immune activation driven by alterations of gut permeability. Hydroxychloroquine (HCQ) reduces endosomal TLR signaling; thus, we verified whether HCQ could dampen immune activation and be associated with an increase in CD4(+) T cells. To this end, we enrolled in a prospective study <em>20</em> HIV-infected immunologic nonresponders (CD4 count < <em>20</em>0 cells/mL or CD4 increase < 5% in the last 12 months) who received 400 mg/day HCQ for 6 months. HCQ had a notable impact on immune activation as shown by significant modifications of the following parameters: (1) reduced plasma lipopolysaccharide; (2) decreased TLR4-expressing CD14(+) cells, TLR4-mediated signal transduction, and mRNA synthesis; (3) reduced percentages of activated CD4(+) (CD4(+)/Ki67(+)) and CD14(+) (CD14(+)/CD69(+)) cells; (4) increased T-regulatory cells (Tregs), naive Tregs, and TLR4-expressing Tregs; (5) augmented plasmacytoid dendritic cells and reduced IFNα-secreting plasmacytoid dendritic cells; and (6) reduced <em>IL</em>-6 and TNFα production. HCQ-induced immune modulation was associated with increased percentages of circulating CD4(+) T cells and was mostly retained 2 months after therapy interruption. HCQ reduces lipopolysaccharide/TLR-mediated immune activation; this compound could be a useful immunomodulant in HIV-infected patients. This study is registered at EutraCT as <em>20</em>09-012499-28 with study number HLS01/<em>20</em>09-1-16-03-<em>20</em>09.

Increased incidence of mortality and morbidity due to cardiopulmonary complications has been found to associate with elevated levels of particulate air pollution (particulate matter with an aerodynamic diameter < 10 microm, PM10 and <2.5 microm, PM2. 5). Lung injury and an imbalance of inflammatory mediators are proposed causative mechanisms, while the toxic constituents may be acidity, transition metals, organic, and biogenic materials. To compare the ability of inhalable fine particles (PM2.5), and coarse particles (PM10-2.5) to cause cell injury and cytokine production in monocytes, dichotomous Andersen samplers were used to collect size-fractionated PM10 for in vitro testing of the particle extracts. Particles from both outdoor and indoor air were collected onto Teflon filters, on nine separate occasions. Each filter was water extracted and each extract assessed for ability to cause cell death, as well as interleukin (<em>IL</em>)-6 and <em>IL</em>-8 production in human monocytes. Significant toxicity and cytokine production was induced by outdoor PM10-2.5, but not by outdoor PM2.5 or the particles collected indoors. Outdoor PM10-2.5 induced <em>20</em> times the amounts of <em>IL</em>-6 and <em>IL</em>-8 than the fine particles. Cytotoxicity was inhibited by deferoxamine, a chelator of transition metals, while cytokine production was not. On the other hand, lipopolysaccharide binding protein (LBP) completely inhibited cytokine induction by PM10-2.5, suggesting that gram-negative bacteria and/or endotoxins are components of PM10-2.5. The effective proinflammatory effects of endotoxin on macrophages may upset lung homeostasis while metals-induced cytotoxicity/necrosis may set up inflammation independent of macrophage-derived cytokines.

Preeclampsia is characterized by hypertension, dyslipidemia, and increased systemic inflammatory response and has been associated with an increased maternal risk of cardiovascular disease later in life. Low-grade chronic inflammation is a risk factor for cardiovascular disease. This study examined changes in inflammatory markers prospectively during pregnancy, the current inflammatory status of women who had a pregnancy complicated by preeclampsia <em>20</em> years previously against matched controls, and the association between inflammatory genes and risk of preeclampsia in a case (n=106) control (n=212) study. In control pregnancies (n=34), mean interleukin-10 (<em>IL</em>-10) levels increased 38% (P=0.012) and tumor necrosis factor-alpha (TNF-alpha) by 33% (P=0.024) between the first and third trimesters. The mean preeclampsia group <em>IL</em>-10 and TNF-alpha rose by 43% (P=0.013 and P=0.0065, respectively) from the first to the third trimester. In women with preeclampsia only, plasma <em>IL</em>-6 increased from the first to the third trimester (1.66 [2.04] to 2.94 [2.47] pg/mL; P=0.0004). Twenty years after the index pregnancy, women who had had preeclampsia demonstrated significantly higher <em>IL</em>-6 to <em>IL</em>-10 ratio (3.96 [6.07] versus 2.12 [1.89]; P=0.034) compared with a healthy index pregnancy <em>20</em> years previously, that persisted after adjustment for smoking and current body mass index. The <em>IL</em>-1beta (C-511T), <em>IL</em>-6 (G-174C), TNF-alpha (G-308A), E-selectin (S128R), intercellular adhesion molecule-1 (K469E), and C-reactive protein (C1059G) polymorphisms were not associated with risk of developing preeclampsia. In conclusion, preeclampsia is associated with short- and long-term changes in inflammatory status.

Cytokines have been proposed as inducers of beta-cell damage in human insulin-dependent diabetes mellitus via the generation of nitric oxide (NO). This concept is mostly based on data obtained in rodent pancreatic islets using heterologous cytokine preparations. The present study examined whether exposure of human pancreatic islets to different cytokines induces NO and impairs beta-cell function. Islets from 30 human pancreata were exposed for 6-144 h to the following human recombinant cytokines, alone or in combination: IFN-gamma (1,000 U/ml), TNF-alpha (1,000 U/ml), <em>IL</em>-6 (25 U/ml), and <em>IL</em>-1 beta (50 U/ml). After 48 h, none of the cytokines alone increased islet nitrite production, but IFN-gamma induced a <em>20</em>% decrease in glucose-induced insulin release. Combinations of cytokines, notably <em>IL</em>-1 beta plus IFN-gamma plus TNF-alpha, induced increased expression of inducible NO synthase mRNA after 6 h and resulted in a fivefold increase in medium nitrite accumulation after 48 h. These cytokines did not impair glucose metabolism or insulin release in response to 16.7 mM glucose, but there was an 80% decrease in islet insulin content. An exposure of 144 h to <em>IL</em>-1 beta plus IFN-gamma plus TNF-alpha increased NO production and decreased both glucose-induced insulin release and insulin content. Inhibitors of NO generation, aminoguanidine or NG-nitro-L-arginine, blocked this cytokine-induced NO generation, but did not prevent the suppressive effect of <em>IL</em>-1 beta plus IFN-gamma plus TNF-alpha on insulin release and content. In conclusion, isolated human islets are more resistant to the suppressive effects of cytokines and NO than isolated rodent islets. Moreover, the present study suggests that NO is not the major mediator of cytokine effects on human islets.

Giant multinucleated cells (GMCs) are associated with granulomatous lesions that form in response to various infectious and noninfectious agents. The present study shows that mouse <em>IL</em>-4 induces the in vitro formation of GMCs by factor-dependent bone marrow and alveolar monocytes via cell fusion. GMCs appear 2 d after incubation of cell cultures with <em>20</em> U/ml or more of <em>IL</em>-4. Anti-<em>IL</em>-4 mAbs block the appearance of GMCs in these cultures, indicating that <em>IL</em>-4 acts directly on monocytes to promote fusion and does not secondarily induce the production of other soluble fusion factors. In soft agar cultures, <em>IL</em>-4 also causes the aggregation of macrophages and diminishes their migration. The role of <em>IL</em>-4 in a granulomatous inflammatory response is discussed.

The purpose of the study was to identify a comprehensive prognostic system of pretreatment clinical parameters in 425 patients (pts) with metastatic renal-cell carcinoma treated with different subcutaneous (s.c.) recombinant cytokine-based home therapies in consecutive trials. Treatment consisted of (A) s.c. interferon-alpha 2a (INF-alpha), s.c. interleukin-2 (<em>IL</em>-2) (n=102 pts), (B) s.c. IFN-alpha 2a, s.c. <em>IL</em>-2, and i.v. 5-fluorouracil (5-FU) (n=235 pts) or (C) s.c. IFN-alpha 2a, s.c. <em>IL</em>-2, and i.v. 5-FU combined with p.o. 13-cis-retinoic acid (13cRA) (n=88 pts). Kaplan-Meier survival analysis, log-rank statistics, and Cox regression analysis were employed to identify risk factors and to create a multiple risk factor model. The following pretreatment risk factors were identified by univariate analysis: (1) three and more metastatic sites, (2) presence of liver, lymph node or bone metastases, (3) neutrophil count>> or = 6500 cells microl(-1), (4) serum lactate dehydrogenase level (LDH)>> or = 2<em>20</em> U l(-1), and (5) serum C-reactive protein level (CRP)>> or = 11 mg l(-1). Cox regression analysis with forward stepwise variable selection identified neutrophil count as the major prognostic factor (hazard ratio=1.9, P<0.001), while serum levels of LDH and CRP, time between diagnosis of tumour and onset of metastatic disease, number of metastatic sites, and bone metastases were significant but somewhat less important prognostic variables within the multiple risk factor model (hazard ratio < or = 1.5). Patients were assigned to one of the three risk groups according to cumulative risk defined as the sum of simplified risk s.c.ores for six pretreatment variables. Low-, intermediate-, and high-risk patients achieved a median overall survival of 32+ months (95% CI 24, 43; 5-year survival of 27%), 18+ months (95% CI 15, <em>20</em>; 5-year survival of 11%), and 8+ months (95% CI 6, 10; 5-year survival of 5%), respectively. These prognostic categories are helpful both in individual patient care and in the assessment of patients entering prospective clinical trials.

Obesity is associated with infiltration of macrophages into adipose tissue. Adipose macrophages may contribute to an elevated inflammatory status by secreting a variety of proinflammatory mediators, including tumor necrosis factor alpha and interleukin-6 (<em>IL</em>-6). Recent data suggest that during diet-induced obesity the phenotype of adipose-resident macrophages changes from alternatively activated macrophages toward a more classical and pro-inflammatory phenotype. Here, we explore the effect of peroxisome proliferator-activated receptor gamma activation on obesity-induced inflammation in 129SV mice fed a high fat diet for <em>20</em> weeks. High fat feeding increased bodyweight gain, adipose tissue mass, and liver triglycerides. Rosiglitazone treatment further increased adipose mass, reduced liver triglycerides, and changed adipose tissue morphology toward smaller adipocytes. Surprisingly, rosiglitazone markedly increased the number of macrophages in adipose tissue, as shown by immunohistochemical analysis and quantification of macrophage marker genes CD68 and F4/80+. In adipose tissue, markers for classically activated macrophages including <em>IL</em>-18 were down-regulated, whereas markers characteristic for alternatively activated macrophages (arginase 1, <em>IL</em>-10) were up-regulated by rosiglitazone. Importantly, conditioned media from rosiglitazone-treated alternatively activated macrophages neutralized the inhibitory effect of macrophages on 3T3-L1 adipocyte differentiation, suggesting that alternatively activated macrophages may be involved in mediating the effects of rosiglitazone on adipose tissue morphology and mass. Our results suggest that short term rosiglitazone treatment increases infiltration of alternatively activated macrophages in adipose tissue. The alternatively activated macrophages might play a role in peroxisome proliferator-activated receptor gamma-dependent expansion and remodeling of adipose tissue.

<em>IL</em>-17 is a T cell-derived cytokine that stimulates stromal cells and macrophages to secrete proinflammatory cytokines. We hypothesized that <em>IL</em>-17 might play a role in alloimmune responses, and that interference with its activity might suppress allograft rejection. <em>IL</em>-17R:Fc or control IgG was added at the start of mouse MLR or was administered i.p. (100-500 microg/day) for different durations post-transplant to murine recipients of MHC-mismatched cardiac allografts. <em>IL</em>-17R:Fc (50-<em>20</em>0 microg/ml) markedly inhibited T cell proliferation in vitro and significantly prolonged nonvascularized cardiac allograft median survival time from 13 to <em>20</em> days (100 microg/day; days 0 and 1) or to 19 days (100-300 microg/day; days 0-4). Survival of vascularized grafts was also extended significantly from 10.5 to 19 days by <em>IL</em>-17R:Fc (500 microg/day; days 0-6). To address a possible mechanism by which <em>IL</em>-17 may promote alloreactivity, we examined the influence of <em>IL</em>-17 on the differentiation and function of bone marrow-derived cells propagated in granulocyte-macrophage CSF with or without <em>IL</em>-4 to promote dendritic cell (DC) growth. A minor proportion of CD11c+ DC expressed the <em>IL</em>-17R. <em>IL</em>-17 promoted the maturation of DC progenitors, as evidenced by increased cell surface expression of CD11c, costimulatory molecules (CD40, CD80, CD86), and MHC class II Ag, and allostimulatory capacity. <em>IL</em>-17 had a lesser effect on the phenotype and function of more fully differentiated myeloid DC. These findings suggest a role for <em>IL</em>-17 in allogeneic T cell proliferation that may be mediated in part via a maturation-inducing effect on DC. <em>IL</em>-17 appears to be a novel target for therapeutic intervention in allograft rejection.

Multicentric Castleman disease (MCD) is a distinct type of lymphoproliferative disorder associated with inflammatory symptoms and interleukin 6 (<em>IL</em>-6) dysregulation. In the context of human immunodeficiency virus (HIV) infection, MCD is associated with Kaposi sarcoma-associated herpesvirus, also called human herpesvirus type 8 (KSHV/HHV8). Within a prospective cohort study on 60 HIV-infected patients with MCD, and a median follow-up period of <em>20</em> months, 14 patients developed KSHV/HHV8-associated non-Hodgkin lymphoma (NHL): 3 "classic" KSHV/HHV8(+) Epstein-Barr virus-positive (EBV(+)) primary effusion lymphoma (PEL), 5 KSHV/HHV8(+) EBV(-) visceral large cell NHL with a PEL-like phenotype, and 6 plasmablastic lymphoma/leukemia (3/3 KSHV/HHV8(+) EBV(-)). The NHL incidence observed in this cohort study (101/1000 patient-years) is about 15-fold what is expected in the general HIV(+) population. MCD-associated KSHV/HHV8(+) NHL fell into 2 groups, suggesting different pathogenesis. The plasmablastic NHL likely represents the expansion of plasmablastic microlymphoma from the MCD lesion and progression toward aggressive NHL. In contrast, the PEL and PEL-like NHL may implicate a different original infected cell whose growth is promoted by the cytokine-rich environment of the MCD lesions.

Dendritic cells (DCs) are the single most central player in all immune responses. To assess whether DC alterations may contribute to the immune dysregulation that affects the elderly, we investigated the effects of ageing on DCs. We analyzed the number, phenotype and function of peripheral blood DCs from 70 healthy subjects aged <em>20</em>-92 years by using flow cytometric methods that allow cell characterization directly in whole blood samples. We demonstrated that the number of myeloid DCs progressively declines with age. This finding was accompanied by a decrease of CD34+ precursors and increase of circulating monocytes, suggesting that the entire differentiation process of antigen presenting cells is partially dysregulated in the elderly. DCs from aged individuals also appeared to have a more mature phenotype and impaired ability to produce <em>IL</em>-12 upon stimulation. These results may help to clarify the contribution of innate immunity to the development of immunosenescence.