OBJECTIVE

From in vitro studies using cultures of orbital fibroblasts, it has become clear that cytokines play an important role in the orbital inflammation in Graves' ophthalmopathy (GO). Orbital fibroblasts seem to be the key target cells of the autoimmune attack, and they are able to express the TSH receptor (TSH-R). In vivo data on the presence of cytokines in orbital tissues are sparse, and mostly limited to samples obtained from patients with endstage, inactive GO; the same holds true for the presence of the TSH-R. The aim of the present study was to determine whether the cytokine profile and TSH-R expression differ in the active vs. the inactive stage of GO.

METHODS

Orbital fat/connective tissue was obtained from six patients with active, untreated GO undergoing emergency orbital decompression, and from 11 patients with inactive GO subjected to rehabilitative decompressive surgery. The mRNA levels of various cytokines and the TSH-R were assessed by real-time polymerase chain reaction (PCR) using the LightCycler. Data are expressed as ratios (unknown mRNA/beta-actin mRNA).

RESULTS

Active GO patients had much higher TSH-R expression than inactive patients: 4/0-24 (median value/range) vs. 0/0-9, P = 0.01. TSH-R expression was related to the Clinical Activity Score (r = 0.595, P = 0.015). Patients with active GO compared to those with inactive GO had higher mRNA levels of the proinflammatory cytokines interleukin-1beta (<em>IL</em>-1beta) (445/153-877 vs. 0/0-455, P = 0.001), <em>IL</em>-6 (1583/968-18825 vs. 559/0-7181, P = 0.01), <em>IL</em>-8 (1422/38-7579 vs. 32/0-1081, P = 0.046) and <em>IL</em>-10 (145/58-318 vs. 27/0-189, P = 0.002). In active GO there also existed a trend towards a predominance of T helper 1 (Th1)-derived cytokines as evident from higher <em>IL</em>-2 (37/0-158 vs. 0/0-68, P = 0.043), interferon-gamma (IFN-gamma) (<em>20</em>/0-79 vs. 0/0-16, P = 0.12) and <em>IL</em>-12 (2.3/0-14.8 vs. 0/0-1.6, P = 0.10) mRNAs. <em>IL</em>-1 receptor agonist (<em>IL</em>-1RA), <em>IL</em>-2 receptor (<em>IL</em>-2R), <em>IL</em>-3, <em>IL</em>-4, <em>IL</em>-5, <em>IL</em>-13, <em>IL</em>-18 and tumour necrosis factor-alpha (TNF-alpha) mRNAs were similar in both groups.

CONCLUSIONS

These data show that at the mRNA level, TSH-R expression is largely present only during the active stages of GO. The active phase is characterized by the presence of proinflammatory and Th1-derived cytokines, whereas other cytokines, among them Th2-derived cytokines, do not seem to be linked to a specific stage of GO.

OBJECTIVE

The ATP-gated P2X(7) receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X(7) receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized.

METHODS

We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1beta release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP).

RESULTS

AZ11645373 up to 10 microM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X(1), rat P2X(2), human P2X(3), rat P2X(2/3), human P2X(4), or human P2X(5) receptors expressed in HEK cells. AZ11645373 inhibited human P2X(7) receptor responses in HEK cells in a non-surmountable manner with K (B) values ranging from 5 - <em>20</em> nM, with mean values not significantly different between assays. K (B) values were not altered by removing extracellular calcium and magnesium. ATP-evoked <em>IL</em>-1beta release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC(50) = 90 nM. AZ11645373 was>> 500-fold less effective at inhibiting rat P2X(7) receptor-mediated currents with less than 50% inhibition occurring at 10 microM.

CONCLUSIONS

AZ11645373 is a highly selective and potent antagonist at human but not rat P2X(7) receptors and will have much practical value in studies of human cells.

Interleukin-6 (<em>IL</em>-6) is one of the major circulating cytokines in catabolic states. To investigate its endocrinologic and metabolic actions in vivo, we studied eight patients with metastatic renal cell cancer two times, once during infusion of saline (control) and once during a 4-h infusion of 150 micrograms recombinant human <em>IL</em>-6 (rh<em>IL</em>-6). Rates of appearance (Ra) of glucose and free fatty acids (FFA) in plasma were measured by using the isotope dilution method. Energy expenditure and substrate oxidation were determined by indirect calorimetry. rh<em>IL</em>-6 induced increases in plasma norepinephrine (+261 +/- 97%, P < 0.001), cortisol (+210 +/- 48%, P < 0.001), and glucagon (+70 +/- 18%, P < 0.001), in resting energy expenditure (+25 +/- 2%, P < 0.001 vs. control), and in plasma FFA concentration (+60 +/- 30%, P < 0.001), FFA Ra (+105 +/- 18%, P < 0.001), and fat oxidation (+38 +/- 16%, P < 0.001). Glucose Ra increased by <em>20</em> +/- 5% (P < 0.01) during rh<em>IL</em>-6 infusion with a concomitant increase in the metabolic clearance rate of glucose. In conclusion, our data demonstrate that rh<em>IL</em>-6 induces many of the endocrinologic and metabolic changes found in catabolic states and thus may mediate some of the metabolic effects previously ascribed to other cytokines.

Expanded genomic information has driven the discovery of new members of the human Class II family of cytokine receptors (CRF2), which now includes 12 proteins. The corresponding cytokines have been identified, paired with their receptors and initially characterized for function. These cytokines include: a new human Type I IFN, IFN-kappa; molecules related to <em>IL</em>-10 (<em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, <em>IL</em>-24, <em>IL</em>-26); and IFN-lambdas (<em>IL</em>-28/29), which have antiviral and cell stimulatory activities reminiscent of Type I IFNs, but act through a distinct receptor. In response to ligand binding, the CRF2 proteins form heterodimers, leading to cytokine-specific cellular responses; these diverse physiological functions are just beginning to be explored. Progress in structural and mutational analysis of ligand-receptor interactions now presents a more reliable framework for understanding receptor-ligand interactions, and for predicting key regions in less well studied members of the CRF2 family. The relationships between the CRF2 proteins will be summarized, as will the progress in identifying patterns of receptor interactions with ligands.

We have tested the possible physical interactions between the iC3b receptor (CR3), lymphocyte function-associated Ag-1, and class III Fc gamma receptor (Fc gamma RIII) at neutrophil surfaces. Cells were labeled using fluorochrome-conjugated Fab or F(ab')2 fragments of antireceptor mAb. Labeled receptors were capped using second-step F(ab')2 fragments of goat anti-mouse Fab antiserum. After <em>20</em> min at 37 degrees C, 68% of the cells capped the anti-CR3 plus second-step complex. Capping was time, temperature, and cytochalasin B sensitive. When capped cells were probed with Fab' or F(ab')2 fragments of anti-Fc gamma RIII labeled with a distinct fluorochrome, 41% of the cells cocapped Fc gamma RIII. Indistinguishable results were obtained when potential antibody combining sites within caps were blocked with a large excess of Fab or F(ab')2 fragments. When Fc gamma RIII was capped, 49% of the cells cocapped CR3. Similarly, LFA-1 cocapped with both CR3 and Fc gamma RIII. Importantly, other membrane components including HLA class I, Mo5, CD13, CR type 1, and <em>IL</em>-8 receptors and N-4-nitrobenzo-2-oxa-1, 3-diszole L-alpha-dimyristoyl phosphatidylethanolamine did not cocap with CR3. However, the positive control Con A did cocap with CR3 and Fc gamma RIII. We next evaluated the effect of saccharides on CR3-Fc gamma RIII cocapping and found that 0.15 M N-acetyl-D-glucosamine (NADG), alpha-methyl-D-mannoside, and D-mannose significantly inhibited cocapping by 70, 58, and 48%, respectively. No inhibition was obtained using glucose, galactose, N-acetyl-neuraminic acid, fucose, sorbitol, fructose, or sucrose. Similarly, Fc gamma RIII-lymphocyte function-associated-1 cocapping was inhibited by NADG. However, the cocapping of CR3 with lymphocyte function-associated-1 or Con A were not affected by 0.15 M NADG, which suggests that NADG inhibition of leukoadhesin-Fc gamma RIII cocapping is not due to a general effect of NADG on capping. Inasmuch as Fc gamma RIII is a glycophospholipid-linked membrane protein, we speculate that it interacts with CR3 and/or lymphocyte function-associated-1 via lectin-like interactions.

We studied the production of the two major mediators of cellular immune responses, Interleukin 1 (<em>IL</em>-1) and Interleukin 2 (<em>IL</em>-2), by the peripheral blood mononuclear cells of 23 burn patients (16 men, seven women, mean age 48.9 years) compared with 23 matched controls (16 men, seven women, mean age 46.7 years). Serial measurements were made of <em>IL</em>-1 production by adherent mononuclear cells after stimulation with lipopolysaccharide and of <em>IL</em>-2 production by lymphocytes after stimulation with phytohemagglutinin (PHA). Eighty determinations of <em>IL</em>-2 production by lymphocytes from 12 patients with greater than 30% body surface area burn revealed a mean <em>IL</em>-2 production of 0.71 u as compared with a mean of 1.23 u for patients with less than 30% burns (p = 0.04). Patients with greater than 30% body surface area burns had significantly reduced <em>IL</em>-2 production (p less than or equal to 0.05) until 60 days after injury, whereas those with smaller burns had reduced <em>IL</em>-2 production only at <em>20</em>-29 and 30-39 days postburn. Nine burn patients with systemic sepsis showed significantly lower <em>IL</em>-2 production (p = 0.03) at 10-29 days postburn than nonseptic patients, and significantly less <em>IL</em>-2 production during septic episodes. Eight patients with greater than 50% suppression of lymphocyte response to PHA produced less <em>IL</em>-2 (0.4 u) than patients with less than 50% suppression, (1.07 u, p = 0.004). <em>IL</em>-1 production was significantly elevated as compared with controls (4.45 u vs. 3.6 u, p = 0.05) early after injury, but was subsequently within the normal range regardless of burn size. The percentage of circulating helper T-lymphocytes, the principal source of <em>IL</em>-2, was also reduced, although this did not always correlate with <em>IL</em>-2 production, which remained depressed after recovery of the helper T-cell population. These results indicate that failure to produce <em>IL</em>-2, a powerful mediator of cellular immune responses, is an important mechanism underlying the defective cell mediated immunity seen in burn patients.

Although cigarette smoking is of paramount importance in the development of chronic obstructive pulmonary disease (COPD), only a small proportion of smokers develop the disease. We tested the hypothesis that the response of the bronchial epithelium to cigarette smoke (CS) differs in patients with COPD. Such a difference might explain in part why only some cigarette smokers develop the disease. We established primary explant cultures of human bronchial epithelial cells (HBEC) from biopsy material obtained from never-smokers who had normal pulmonary function, smokers with normal pulmonary function, and smokers with COPD, and exposed these for <em>20</em> min to CS or air. Measurements were subsequently made over a period of 24 h of transepithelial permeability and release of interleukin (<em>IL</em>)-1beta and soluble intercellular adhesion molecule-1 (sICAM-1). In addition, intracellular reduced glutathione (GSH) levels were measured after 24 h incubation. Exposure to CS increased the permeability of these cultures in all study groups, but the most marked effect was observed in cultures from patients with COPD (mean increase, 85.5%). The smallest CS-induced increase in the permeability was observed in HBEC cultured from smokers with normal pulmonary function (mean, 25.0%), and this was significantly lower than that of HBEC from never-smokers (mean, 53.4%) (P<0.001). Compared with exposure to air, exposure to CS led to a significantly increased release of these mediators from cultures of the never-smoker group (mean 250.0% increase in <em>IL</em>-1beta and mean 175.3% increase in sICAM-1 24 h after exposure) and COPD group (mean 383.3% increase in <em>IL</em>-1beta and mean 97.4% increase in sICAM-1 24 h after exposure). In contrast, CS exposure did not influence significantly the release of either mediator from the cells of smokers with normal pulmonary function. Levels of intracellular GSH were significantly higher in cultures of HBEC derived from smokers, both those with normal pulmonary function and those with COPD, compared with cultures from healthy never-smokers. Exposure to CS significantly decreased the concentration of intracellular GSH in all cultures. However, the fall in intracellular GSH was significantly greater in cells from patients with COPD (mean 72.9% decrease) than in cells from never-smokers (mean 61.4% decrease; P = 0.048) or smokers with normal pulmonary function (mean 43.9% decrease; P = 0.02). These results suggest that whereas smokers with or without COPD demonstrate increased levels of GSH within bronchial epithelial cell cultures, those with COPD have a greater susceptibility to the effects of CS in reducing GSH levels and causing increased permeability and release of proinflammatory mediators such as <em>IL</em>-1beta and sICAM-1.

OBJECTIVE

In order to evaluate the possible antiinflammatory action of bisphosphonates, the effect of the drugs on the secretion of proinflammatory cytokines (IL-1 beta, IL-6 and TNF alpha) from macrophages was studied. Liposomes or high concentration of extracellular calcium was used to enhance the intracellular delivery of bisphosphonates.

METHODS

RAW 264 cells were used as macrophage model, and they were induced with lipopolysaccharide to produce the cytokines. The cytokine concentrations in the culture supernatants were measured with time-resolved fluoroimmunoassay.

RESULTS

As a free drug, clodronate and pamidronate, but not etidronate, inhibited LPS-stimulated secretion of the cytokines from macrophage-like RAW 264 cells. Low concentrations of pamidronate, however, induced the IL-6 secretion, and the cytokine inhibitory action at the higher concentrations of pamidronate was attributed to cytotoxicity of the compound. The cytokine induction or toxic effects were not observed with clodronate or etidronate. When the drugs were encapsulated in negatively charged unilamellar liposomes, the inhibitory potency of both clodronate and etidronate enhanced by a factor of 10-20, while that of pamidronate was not increased. The complex formation of bisphosphonates with extracellular calcium, although enhancing the uptake of the compounds by macrophages, did not considerably increase their cytokine inhibitory potency.

CONCLUSIONS

Bisphosphonates have inhibitory action on cytokine secretion by macrophages. The non-cytotoxic cytokine inhibition by liposome encapsulated clodronate could be beneficial in local inflammatory diseases, where the inflammation is sustained by the excessive amounts of inflammatory cytokines produced by activated macrophages.

The regulation of human interleukin 2 (<em>IL</em>-2) mRNA production in induced normal lymphocytes was studied by following the expression of isolated mRNA in microinjected oocytes of Xenopus laevis. Mitogenic stimulation results in the appearance of greatly increased levels of <em>IL</em>-2 mRNA activity. This process requires de novo transcription. Induction is followed promptly by a shutoff of active <em>IL</em>-2 mRNA formation. This shutoff requires the synthesis of a protein repressor and can be prevented by cycloheximide, an inhibitor of translation. The presence of cycloheximide leads to extensive superinduction of <em>IL</em>-2, concomitant with an increase in active <em>IL</em>-2 mRNA formation up to 30-fold over normal levels. The repressor appears to be short-lived, as the addition of cycloheximide after shutoff leads to an immediate resumption of active <em>IL</em>-2 mRNA formation. The shutoff mechanism is restored rapidly upon removal of cycloheximide. The repressed state is readily reversed also by reinduction of the cells, even soon after shutoff has occurred, without a refractory period. The accumulated active <em>IL</em>-2 mRNA decays with a half-life of about <em>20</em> hr. The net result is the generation of a relatively short wave of <em>IL</em>-2 mRNA activity, demonstrating the tight control of <em>IL</em>-2 gene expression.

CD4(+)CD25(+) T cells play a central role in the suppression of autoimmunity and inflammation, making their in vivo expansion a highly attractive therapeutic target. By phenotyping with a novel rat CTL antigen-4 (CTLA-4)-specific monoclonal antibody (mAb) and functional in vitro assays, we here first establish that rat CD4(+)CD25(+) T cells correspond to the regulatory T cells (Treg cells) described in mice and humans: they constitutively express CTLA-4, produce <em>IL</em>-10 but not <em>IL</em>-2, and are able to suppress the proliferation of costimulated CD25-negative indicator cells. Furthermore, we show that rat Treg cells respond less well than CD25(-) T cells to conventional costimulation, but are readily expanded in vitro with "superagonistic" CD28-specific mAb which are potent mitogens for all T cells without the need for TCR engagement. In vivo, functional Treg cells are preferentially expanded by CD28 stimulation over other T cell subsets, leading to a <em>20</em>-fold increase within 3 days in response to a single antibody dose. These data suggest that CD28-driven activation of Treg cells may be highly effective in the treatment of inflammatory and autoimmune diseases.

The importance of IgE in airway inflammation and development of AHR in allergen-sensitized mice has been compared and contrasted in different models of sensitization and challenge. Using different modes of sensitization in normal and genetically manipulated mice after anti-IgE treatment, we have been able to distinguish the role of IgE under these different conditions. Striking differences in the three sensitization protocols were delineated in terms of the role of allergen-specific IgE, extent of eosinophilic airway inflammation, and development of AHR (Table 1). The highest levels of IgE and eosinophil infiltration (approximately <em>20</em>-fold increases) were achieved after systemic sensitization with allergen (plus adjuvant) followed by repeated airway challenge. Passive sensitization with allergen-specific IgE followed by limited airway challenge induced a modest eosinophilic inflammatory response in the airways despite high levels of serum IgE. Exposure to allergen exclusively via the airways also resulted in a modest serum IgE response and a limited eosinophilic inflammatory response (approximately fourfold increases). Under all of these conditions, inhibition of <em>IL</em>-5-mediated eosinophilic airway inflammation was associated with attenuation of AHR. In contrast, the differences in the responses to the different modes of allergen exposure were associated with differences in the requirements for IgE in the development of AHR (Table 1). In the two models associated with mild eosinophil infiltration (passive sensitization and exclusive airway exposure), IgE was required for the development of AHR but did not substantially enhance airway inflammation on its own. However, IgE-allergen interaction was able to enhance T-cell function in vitro and induce T-cell expansion in vivo. In mice systemically sensitized and challenged via the airways, IgE (or IgE-mediated mast-cell activation) was not required for T-cell activation, eosinophilic inflammation and activation in the airways, or development of AHR. This was most clearly seen in B-cell-deficient and mast-cell-deficient, low-IgE-responder mouse strains (B6, B10) and in anti-IgE-treated high-IgEresponder mice (BALB/c). At the same time, we confirmed the importance of IgE in the induction of immediate-type hypersensitivity (mast-cell activation, immediate cutaneous hypersensitivity, passive cutaneous and systemic anaphylaxis). These differences were also highlighted by the means used to detect altered airway function. Passive sensitization and limited airway challenge or exclusive airway exposure to allergen over 10 days elicited changes in airway function that could be detected only in tracheal smooth-muscle preparations exposed to EFS. In contrast, systemic sensitization followed by repeated airway challenge resulted not only in changes in the contractile response to EFS but also in increased responsiveness to inhaled MCh. Thus, these results distinguish not only the differential involvement of IgE and eosinophil numbers but also their contribution to the readouts used to monitor airway function. Based on these studies, we conclude that IgE plays an important role in the development of airway inflammation and AHR under conditions in which limited <em>IL</em>-5-mediated eosinophilic airway infiltration is induced. In conditions where a robust eosinophilic inflammation of the airways is elicited, IgE (and IgE-mediated mast-cell activation) does not appear to be essential for airway inflammation and the development of AHR, detected as increased responsiveness to inhaled MCh. These findings reveal the potential importance of differential targeting in the treatment of allergic diseases with a predominance of IgE-mediated symptoms, e.g., allergic rhinitis and conjunctivitis, where anti-IgE may be an effective therapy, compared to those diseases with a predominant inflammatory component, e.g., AHR in atopic bronchial asthma, where anti-inflammatory or anti-<em>IL</em>-5 therapy may be more beneficial.

We have isolated the mouse interleukin 3 (<em>IL</em>-3) gene from a mouse sperm DNA library based on homology with the mouse mast-cell growth-factor (MCGF) cDNA sequence. The nucleotide sequence determined for the gene and its flanking regions reveals that the <em>IL</em>-3 gene is composed of four introns and five exons. The nucleotide sequence of the exons agrees with that determined for the MCGF cDNA. A "TATA"-like sequence preceded by a G + C-rich region is found in the 5' flanking region. At the 3' region of the second intron are nine repeats of a closely related 14-base-pair (bp) sequence. These repeated sequences share extensive homology with a <em>20</em>-bp repeated sequence found in the human genome, which was shown to have enhancer activity. Eight out of nine repeats form a 73-bp duplicated sequence and each 73-bp repeat contains sequences homologous to the core sequence suggested for enhancer elements. These sequences may play a role in the expression of the <em>IL</em>-3 gene in concanavalin A- or antigen-stimulated T lymphocytes. Stable transformants of L cells generated by co-transformation of the <em>IL</em>-3 gene with pSV2neo constitutively express MCGF activity indicating that the isolated gene is functional in vivo.

The alveolar macrophage (AMO) in its pivotal position for pulmonary host defense may play a prominent role in the orchestration of polymorphonuclear leukocyte (PMN) diapedesis. We demonstrate that the human AMO may participate in these inflammatory events through the production of a novel neutrophil chemotactic factor, interleukin-8 (<em>IL</em>-8). The induction of AMO-derived <em>IL</em>-8 by tumor necrosis factor (TNF), lipopolysaccharide (LPS), and interleukin-1 (<em>IL</em>-1 beta) was shown to be both dose and time dependent. Maximal <em>IL</em>-8 gene expression, as assessed by Northern blot analyses, was achieved with <em>20</em> ng/ml and 1 microgram/ml, respectively, for each of the cytokines and LPS. A kinetic study of TNF-, <em>IL</em>-1 beta-, and LPS-treated AMOs showed significant steady-state <em>IL</em>-8 mRNA accumulation post-stimulation at 1 h, peaking by 8 h, with a decline over the next 16 h. Immunohistochemical staining using rabbit anti-human <em>IL</em>-8 antibody demonstrated significant immunolocalization of cell-associated <em>IL</em>-8 antigen at 4 h, with persistence over the next <em>20</em> h. Chemotactic bioactivity peaked by 8 h, with continued production over the next 16 h. Chemotactic bioactivity from AMO-conditioned media was inhibited by <em>IL</em>-8 antiserum by 2, 31, 44, and 47%, respectively, for unstimulated control, LPS-, <em>IL</em>-1 beta-, and TNF-treated cells. Preimmune serum had no effect on chemotactic activity. These data support the central role of the AMO in the elicitation of PMNs into the lung via the production of <em>IL</em>-8.

The novel hematopoietic growth factor FLT3 ligand (FL) is the cognate ligand for the FLT3, tyrosine kinase receptor (R), also referred to as FLK-2 and STK-1. The FLT3R belongs to a family of receptor tyrosine kinases involved in hematopoiesis that also includes KIT, the receptor for SCF (stem cell factor), and FMS. the receptor for M-CSF (macrophage colony- stimulating factor). Restricted FLT3R expression was seen on human and murine hematopoietic progenitor cells. In functional assays recombinant FL stimulated the proliferation and colony formation of human hematopoietic progenitor cells, i.e. CD34+ cord and peripheral blood, bone marrow and fetal liver cells. Synergy was reported for co-stimulation with G-CSF (granulocyte-CSF). GM-CSF (granulocyte-macrophage CSF), M-CSF, interleukin-3 (<em>IL</em>-3), PIXY-321 (an <em>IL</em>-3/GM-CSF fusion protein) and SCF. In the mouse, FL potently enhanced growth of various types of progenitor/precursor cells in synergy with G-CSF, GM-CSF, M-CSF, <em>IL</em>-3, <em>IL</em>-6, <em>IL</em>-7, <em>IL</em>-11, <em>IL</em>-12 and SCF. The well-documented involvement of this ligand-receptor pair in physiological hematopoiesis brought forth the question whether FLT3R and FL might also have a role in the pathobiology of leukemia. At the mRNA level FLT3R was expressed by most (80-100%) cases of AML (acute myeloid leukemia) throughout the different morphological subtypes (MO-M7), of ALL(acute lymphoblastic leukemia) of the immunological subtypes T-ALL and BCP-ALL (B cell precursor ALL including pre-pre B-ALL, cALL and pre B-ALL), of AMLL (acute mixed-lineage leukemia), and of CML (chronic myeloid leukemia) in lymphoid or mixed blast crisis. Analysis of cell surface expression of FLT3R by flow cytometry confirmed these observations for AML (66% positivity when the data from all studies are combined), BCP-ALL (64%) and CML lymphoid blast crisis (86%) whereas less than 30% of T-ALL were FLT3R+. The myeloid, monocytic and pre B cell type categories also contained the highest proportions of FLT3R+ leukemia cell lines . In contrast to the selective expression of the receptor, FL expression was detected in 90-100% of the various cell types of leukemia cell lines from all hematopoietic cell lineages. The potential of FL to induce proliferation of leukemia cells in vitro was also examined in primary and continuously cultured leukemia cells. The data on FL-stimulated leukemia cell growth underline the extensive heterogeneity of primary AML and ALL samples in terms of cytokine-inducible DNA synthesis that has been seen with other effective cytokines. While the majority of T-ALL (0-33% of the cases responded proliferatively; mean 11%) and BCP-ALL (0-30%; mean <em>20</em>%) failed to proliferate in the presence of FL despite strong expression of surface FLT3R, FL caused a proliferative response in a significantly higher percentage of AML cases (22-90%; mean 53%). In the panel of leukemia cell lines examined only myeloid and monocytic growth factor- dependent cell lines increased their proliferation upon incubation with FL, whereas all growth factor-independent cell lines were refractory to stimulation. Combinations of FL with G-CSF, GM-CSF, M-CSF, <em>IL</em>-3, PIXY- 321 or SCF and FL with <em>IL</em>-3 or <em>IL</em>-7 had synergistic or additive mitogenic effects on primary AML and ALL cells, respectively. The potent stimulation of the myelomonocytic cell lines was further augmented by addition of bFGF (basic fibroblast growth factor), GM-CSF, <em>IL</em>-3 or SCF. The inhibitory effects of TGF-beta 1 (transforming growth factor-beta 1) on FL- supported proliferation were abrogated by bFGF. Taken together, these results demonstrate the expression of functional FLT3R capable of mediating FL- dependent mitogenic signaling in a subset of AML and ALL cases further underline the heterogeneity of AML and ALL samples in their proliferative response to cytokine.

Inflammation is emerging as an important mechanism for micro- and macrovascular complication of diabetes. The macrophage plays a key role in the chronic inflammatory response in part by generating particular cytokines. <em>IL</em>-1beta, <em>IL</em>-6, <em>IL</em>12, <em>IL</em>-18, TNFalpha, and interferon-gamma are produced primarily in macrophages and have been associated with accelerated atherosclerosis and altered vascular wall function. In this study, we evaluated the effect and mechanism of high glucose (HG) on gene expression of these cytokines in mouse peritoneal macrophages (MPM). HG led to a 2-fold increase in the mRNA expression of these cytokines, with <em>IL</em>-12 showing the highest activation (5.4-fold) in a time-dependent (3-12 h) and dose-dependent (10, 17.5, and 25 mmol/liter) manner. The effects were specific to HG because mannitol and 3-O-methyl-glucose had no effect on cytokine mRNA expression. HG also increased <em>IL</em>-12 protein accumulation from MPM. We also explored the role of induced and spontaneous diabetes on inflammatory cytokine expression in MPM. Increases in expression in MPM of multiple inflammatory cytokines, including a <em>20</em>-fold increase in <em>IL</em>-12 mRNA, were observed in streptozotocin-induced type 1 diabetic mice as well as type 2 diabetic db/db mice, suggesting that cytokine gene expression is increased by hyperglycemia in vivo. We next explored potential mechanisms of HG-induced increases in <em>IL</em>-12 mRNA. HG increased the activity of protein kinase C, p38 MAPK (p38), c-Jun terminal kinase, and inhibitory-kappaB kinase in MPM. Furthermore, inhibitors of these signaling pathways significantly reduced HG-induced <em>IL</em>-12 mRNA expression in MPM. These results provide evidence for a potentially important mechanism linking elevated glucose and diabetes to inflammation.

Using the murine colon adenocarcinoma C-26 cell line, engineered to release granulocyte colony-stimulating factor (G-CSF) (C-26/G-CSF), were studied the mechanisms responsible for inhibition of tumor take in syngeneic animals and of regression of an established tumor in sublethally irradiated mice injected with these cells. Immunocytochemistry and in situ hybridization, performed to characterize tumor-infiltrating leukocytes and their cytokine expression, respectively, indicated that polymorphonuclear leukocytes (PMN) were the major cells responsible for inhibition of tumor take and that they expressed mRNA for interleukin 1 alpha (<em>IL</em>-1 alpha), <em>IL</em>-1 beta, and tumor necrosis factor alpha (TNF-alpha). Expression of interferon gamma (IFN-gamma) and of <em>IL</em>-4 was undetectable, consistent with the absence of T lymphocytes at the site of tumor injection. In mice injected with C-26/G-CSF cells after 600-rad irradiation, the tumors grew to approximately 1.5 cm in 30 d, regressing completely thereafter in 70-80% of mice. During the growing phase, tumors were infiltrated first by PMN (between days 15 and <em>20</em>), then by macrophages, and last by T lymphocytes. Both CD4+ and CD8+ T cells were present but only CD8 depletion significantly abrogated tumor regression. Depletion of PMN by the RB6-8C5 antigranulocytes monoclonal antibody reduced the number of T cells infiltrating the tumor and prevented tumor regression. In situ hybridization performed at the beginning of tumor regression revealed the presence of mRNA for <em>IL</em>-1 alpha, <em>IL</em>-1 beta, and TNF-alpha, but also the presence of cells, with lymphoid morphology, expressing IFN-gamma. Tumors from mice treated with recombinant IFN-gamma (between days <em>20</em> and 35) were rejected faster, whereas mice treated with antibodies to IFN-gamma (from day <em>20</em>) died of progressive tumor. Cyclosporin A treatment (started at day <em>20</em>) also abrogated tumor regression. These results indicate that inhibition of tumor take and regression in this model occurs through different mechanisms that involve PMN and PMN-T cell interactions, respectively, as well as a combination of cytokines that, for tumor regression, require IFN-gamma. Thus, gene transfer of a single cytokine gene such as G-CSF into tumor cells appears to be sufficient to trigger the cascade of cell interactions and cytokine production necessary to destroy a cancer nodule.

BACKGROUND

Vulnerable plaque demonstrates intense inflammation in which macrophages secrete matrix metalloproteinases (MMPs) that degrade the fibrous cap, ultimately leading to rupture, in situ thrombosis, and an associated clinical event. Thus, inhibition of MMP activity or more general suppression of vascular inflammation are attractive targets for interventions intended to reduce plaque rupture. We hypothesized that subantimicrobial doses of doxycycline (SDD) (<em>20</em> mg twice daily) would benefit patients with coronary artery disease by reducing inflammation and MMP activity and thus possibly prevent coronary plaque rupture events.

RESULTS

We conducted a prospective, randomized, double-blind, placebo-controlled pilot study of 6 months of SDD or placebo treatment to reduce inflammation and prevent plaque rupture events. A total of 50 patients were enrolled, of whom 24 were randomized to placebo and 26 to SDD. At 6 months, there was no difference in the composite endpoint of sudden death, fatal myocardial infarction (MI), non-fatal MI, or troponin-positive unstable angina in SDD compared with placebo-treated patients (8.4% versus 0%, P=0.491). Biochemical markers of inflammation were assessed in plasma at study entry and after 6 months of therapy in 30 patients. In SDD-treated patients, high-sensitivity C-reactive protein (CRP) was reduced by 46% from 4.8+/-0.6 microg/mL to 2.6+/-0.4 microg/mL (P=0.007), whereas CRP was not significantly reduced in placebo patients. Interleukin (IL)-6 decreased from 22.1+/-3.7 pg/mL at baseline to 14.7+/-1.8 pg/mL at 6 months in SDD-treated patients (P=0.025) but did not decrease significantly in placebo-treated patients. On zymography, pro-MMP-9 activity was reduced 50% by SDD therapy (P=0.011), whereas it was unchanged by placebo treatment.

CONCLUSIONS

SDD appears to exert potentially beneficial effects on inflammation that could promote plaque stability. These findings should be investigated in a larger study.

BACKGROUND

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by a wide variety of autoantibodies, some of which are pathogenic. In recent years it has become more evident that the polyclonal B cell activation in SLE is T-cell dependent. The stimulation of the autoantibody producing B cells is likely mediated by the TH2 subtype of T cells producing IL-4, IL-5, IL-6 and IL- 10, whereas the TH1 subtype secreting IL-2 and IFN-gamma predominates in cell-mediated immune response. Tumor Necrosis Factor (TNF-alpha) is both a proinflammatory and an immunoregulatory cytokine. TNF-alpha has differential effects on B cells, on T cells and on dendritic cells as well as on the process of programmed cell death. Understanding how the immune system integrates the pleiotropic properties of TNF-alpha is a challenge, particularly so in diseases like SLE. Meanwhile the role of IL-6 in the pathogenesis of SLE is controversial.

OBJECTIVE

To investigate whether serum levels of TNF-alpha and IL-6 is higher in Egyptian patients with SLE than healthy control volunteers and its correlation with the clinical activity in patients with different activity scores as measured by Systemic Lupus Erythmatosus Disease Activity Index (SLEADI).

METHODS

Sixty individuals (40 patients with Systemic lupus Erythmatosus and 20 healthy control volunteers) were the subject of this study, they were subjected to thorough clinical examination, laboratory investigations, their clinical disease activity was scored according to SLEDAI, and serum sampling was obtained for TNF-alpha and IL-6 levels assay. Renal biopsy was carried out and examined by light microscopy by a pathologist blinded with the clinical activity.

RESULTS

The mean level of TNF-alpha was (766.95+/-357.82Pg/ml) for patients with active disease while it was (314.01+/-100.87Pg/ml) for those with inactive disease and (172.7+/-39.19Pg/ml) for the healthy control group. The difference was statistically significant (P=.002). The mean level of IL-6 was (135.4+/-54.23Pg/ml) for patients with active disease while it was (47.33+/-18.61Pg/ml) for those with inactive disease and (21.15+/-10.99Pg/ml) for the healthy control group. The difference was statistically significant (P=.002). A significant correlations between TNF-alpha and IL-6 serum levels and the SLEDAI score was observed (r=.743 and .772, respectively).

CONCLUSIONS

Serum TNF-alpha and IL-6 are sensitive markers of SLE disease activity. They may be useful independent markers for prediction of SLE disease activity and to differentiate normal subjects from those having SLE. Possible therapeutic implications in the treatment of SLE in the future deserve wide scale trials.

Presence of the Helicobacter pylori adherence factor blood group Ag-binding adhesin (BabA; binding to Lewis(b) (Le(b))) is associated with ulcer disease, adenocarcinoma, and precancerous lesions. The importance of BabA for bacterial colonization and the inflammatory response is unknown. A total of 141 antral biopsies from H. pylori-infected patients were assessed in regard to the degree of granulocytic (G0 degrees--G3 degrees) and lymphocytic (L1 degrees--L3 degrees) infiltration. DNA genotypes of babA2 (the transcriptionally active gene of BabA), cagA, and vacAs1/2 were determined by PCR. Colonization density and Le(b) status on gastric epithelial cells were determined by immunohistochemistry. Real-time quantitative (TaqMan) RT-PCR determined mRNA expression of <em>IL</em>-8, TNF -alpha, and the Th1 markers IFN-gamma and the <em>IL</em>-12R beta2 chain. A total of 91% of infected patients were Le(b) positive. The vacAs1(+)/cagA(+) strains harboring babA2 showed significantly higher levels of granulocytic infiltration, bacterial colonization, and <em>IL</em>-8 mRNA than vacAs1(+)/cagA(+) strains lacking babA2. <em>IL</em>-8 mRNA and protein production by KATO III cells in vitro increased dose dependently with addition of different numbers of type 1 strains (G27 and 2808 strains, 0.1--<em>20</em> bacteria/cell). The mRNA expression of TNF-alpha, IFN-gamma, and <em>IL</em>-12R beta2 was higher in H. pylori-positive patients than in controls, but it did not differ significantly between patients infected with different strain types. These data suggest that BabA facilitates colonization of H. pylori and thereby increases <em>IL</em>-8 response, resulting in enhanced mucosal inflammation. Infection with strains harboring BabA thereby augment a nonspecific immune response, whereas the Th1 response toward H. pylori appears to be independent of BabA, cytotoxin-associated gene A, or vacuolating cytotoxin.

<em>IL</em>-19 belongs to the <em>IL</em>-10 family, which includes <em>IL</em>-10, <em>IL</em>-19, <em>IL</em>-<em>20</em>, <em>IL</em>-22, melanoma differentiation-associated gene-7 (<em>IL</em>-24), and AK155 (<em>IL</em>-26). <em>IL</em>-10 has been shown to inhibit allergen-induced airway hyperreactivity and inflammation. To determine whether <em>IL</em>-19 was also associated with asthma, we used ELISA to analyze the serum level of <em>IL</em>-19 in patients with asthma and found that their serum <em>IL</em>-19 levels were twice those of healthy controls. Patients with a high level of <em>IL</em>-19 also had high levels of <em>IL</em>-4 and <em>IL</em>-13. In a dust mite-induced murine model of asthma, we found that <em>IL</em>-19 level in asthmatic BALB/cJ mice was also twice that of healthy control mice. <em>IL</em>-19 transcript was also induced in the lungs of asthmatic mice. Electroporation i.m. of the <em>IL</em>-19 gene into healthy mice up-regulated <em>IL</em>-4 and <em>IL</em>-5, but not <em>IL</em>-13. However, <em>IL</em>-19 up-regulated <em>IL</em>-13 in asthmatic mice. In vitro, <em>IL</em>-19 induced <em>IL</em>-4, <em>IL</em>-5, <em>IL</em>-10, and <em>IL</em>-13 production by activated T cells. Activation of T cells was required for induction of <em>IL</em>-13 because <em>IL</em>-19 did not induce <em>IL</em>-13 production on nonstimulated T cells. Taken together, these results demonstrated that <em>IL</em>-19 up-regulates Th2 cytokines on activated T cells and might play an important role in the pathogenesis of asthma.

BACKGROUND

trophoblast cells have been demonstrated to regulate monocyte migration and differentiation, leading to pro-inflammatory profiles. Because trophoblast cells release exosomes with immunoregulatory properties, trophoblast-derived exosomes are proposed to 'educate' monocytes, creating a pro-inflammatory environment.

METHODS

exosomes were isolated from conditioned media of Swan71 cells by ultrafiltration and ultracentrifugation. Exosome-induced migration was assessed using a two-chamber system. Cytokine profiles were defined using cytokine arrays, and mRNA levels of affected cytokines were examined by qRT-PCR and ELISA.

RESULTS

within <em>20</em> min, 8-10% of monocytes took up labeled exosomes isolated from Swan71 cells. Trophoblast-derived exosomes increased monocyte migration in a dose-dependent manner and produced significant increases in production of interleukin (<em>IL</em>)-1β, <em>IL</em>-6, Serpin-E1, granulocyte colony-stimulating factor, granulocyte/monocyte colony-stimulating factor, and tumor necrosis factor-α.

CONCLUSIONS

this study presents the initial demonstration that trophoblast-derived exosomes are capable of recruiting and 'educating' monocytes to produce pro-inflammatory cytokine/chemokine profiles in a cell-contact-independent manner.

Interleukin (<em>IL</em>)-13 is produced by T helper 2 (Th2)-type cells and inhibits the production of proinflammatory cytokines by activated monocytes, while <em>IL</em>-18 is a pleiotropic cytokine that induces interferon-gamma and plays an important role in the development of Th1-type cells. Role of the shift from a Th1-type response to Th2-type has been suggested in the pathogenesis of dengue hemorrhagic fever (DHF). This study was undertaken to investigate the possible protective/pathogenic role of <em>IL</em>-13 and <em>IL</em>-18 in patients with DHF. Sera were collected from a total of 84 patients with various grades of dengue illness and 21 normal healthy controls and tested for <em>IL</em>-13 and <em>IL</em>-18 levels using commercial enzyme-linked immunosorbent assay kits. The results showed that very low levels of <em>IL</em>-13 (4+/-3 pg ml(-1)) and <em>IL</em>-18 (15+/-4 pg ml(-1)) were detected in the sera of healthy controls. In dengue patients, the levels of <em>IL</em>-13 and <em>IL</em>-18 were the highest in the patients with DHF grade IV (<em>20</em>5+/-103 pg ml(-1) and 366+/-155 pg ml(-1), respectively) and the lowest in patients with dengue fever (22+/-12 pg ml(-1) and 76+/-50 pg ml(-1), respectively). Both the cytokines appeared (<em>IL</em>-13=<em>20</em>+/-11 pg ml(-1) and <em>IL</em>-18=70+/-45 pg ml(-1)) during the first 4 days of illness and reached peak levels (<em>IL</em>-13=<em>20</em>4+/-96 pg ml(-1) and <em>IL</em>-18=360+/-148 pg ml(-1)) by day 9 onwards. The presence of high levels of <em>IL</em>-13 and <em>IL</em>-18 during severe illness and late phases of the disease suggests that both of these cytokines may contribute to the shift from a Th1- to Th2-type response and thus to the pathogenesis of DHF.

BACKGROUND

Atopic dermatitis (AD) is an inflammatory skin disease affecting 10% to <em>20</em>% of children and 1% to 3% of adults in industrialized countries. Enhanced expression of <em>IL</em>-31 is detected in skin samples of patients with AD, but its physiological relevance is not known.

OBJECTIVE

We sought to determine the role of IL-31 in skin differentiation.

METHODS

We used human 3-dimensional organotypic skin models with either primary keratinocytes or HaCaT keratinocytes with inducible IL-31 receptor α to evaluate the effect of IL-31. The consequences were studied by using histology, the expression of markers analyzed by immunofluoresence and quantitative RT-PCR, and gene expression arrays.

RESULTS

We observed that <em>IL</em>-31 interferes with keratinocyte differentiation. Gene expression analysis revealed a limited set of genes deregulated in response to <em>IL</em>-31, including <em>IL</em><em>20</em> and <em>IL</em>24. In HaCaT keratinocytes with inducible <em>IL</em>-31 receptor α, <em>IL</em>-31 inhibited proliferation upon induction of <em>IL</em>-31 receptor α by inducing cell cycle arrest. As in primary cells, <em>IL</em>-31-treated HaCaT cells elicited a differentiation defect in organotypic skin models, associated with reduced epidermal thickness, disturbed epidermal constitution, altered alignment of the stratum basale, and poor development of the stratum granulosum. The differentiation defect was associated with a profound repression of terminal differentiation markers, including filaggrin, an essential factor for skin barrier formation, and a reduced lipid envelope. The highly induced proinflammatory cytokines <em>IL</em>-<em>20</em> and <em>IL</em>-24 were responsible for part of the effect on FLG expression and thus for terminal differentiation.

CONCLUSIONS

Our study suggests that IL-31 is an important regulator of keratinocyte differentiation and demonstrates a link between the presence of IL-31 in skin, as found in patients with AD, and filaggrin expression.

Serum interleukin 6 (<em>IL</em>-6) levels were measured in 75 patients with lung cancer and in <em>20</em> patients with benign lung diseases. <em>IL</em>-6 was detectable in 29 patients with lung cancer (39%), but was not detectable in any of the patients with benign lung diseases. Serum C-reactive protein levels and plasma fibrinogen levels were significantly higher and serum albumin concentration was significantly lower in lung cancer patients with detectable serum <em>IL</em>-6 levels than in those without detectable serum <em>IL</em>-6 levels and in patients with benign lung diseases. On the other hand, no significant difference was observed in blood platelet counts in these three groups. Moreover, serum <em>IL</em>-6 levels were not significantly different in lung cancer patients with or without clinically demonstrated distant metastasis. These results suggest that <em>IL</em>-6 may be a mediator of various reactions including an inflammatory response in lung cancer patients.