Dexamethasone suppresses gene expression and production of IL-13 by human mast cell line and lung mast cells

2,4-Dichlorophenoxybutyric acid (2,4-DB) is principally used in the United States as a herbicide on peanuts, soybeans, and alfalfa. In Europe, it is used on cereals, undersown cereals, lucerne (alfalfa), clover, and clover mixtures. A 1-year chronic toxicity study in the dog was performed on 2,4-DB. Doses in the study of 0, 75, 225, and 450 ppm were administered to six animals/sex/group. The top dose was reduced from 675 ppm during week 7 of the study due to body weight loss and decreased food consumption. Four animals/sex/group were euthanized after 52 weeks of treatment and two animals/sex/group were placed on control diet for 4 weeks and euthanized at week 56. Treatment-related findings included reductions in body weight gain and food consumption, and minor increases in inorganic phosphorus, blood urea nitrogen, creatinine, aspartate aminotransferase, and alanine aminotransferase. After the 4-week recovery period, the only parameter that did not return to control levels was the aspartate aminotransferase. Gross pathology evaluation noted distended gallbladders and decreased organ weights were noted in females for the adrenal, spleen, and ovaries. Histologically, the liver and kidney were the target organs. The data from the study support a chronic no observed adverse effect level of 75 ppm (2.39 and 2.15 mg/kg/day for males and females, respectively) for 2,4-DB. There was no indication of any immunotoxic or oncogenic response in the studies. In conclusion, the findings in this study indicate the general low toxicity of 2,4-DB following chronic dietary exposure in the dog.     

Stem cell factor-induced downregulation of c-kit in human lung mast cells and HMC-1 mast cells

Recent data suggest that local overexpression of the tissue-hormone c-kit ligand (stem cell factor [SCF]) is associated with accumulation of mast cells (MCs) and a decrease in expression of c-kit in the accumulated MCs [28]. In the present study, the effects of recombinant human (rh) SCF on expression of c-kit mRNA and c-kit protein in isolated human MCs and a human mast cell line, HMC-1, were analyzed. Incubation of isolated lung MC with rhSCF (100 ng/mL) for 120 minutes resulted in decreased expression of c-kit mRNA (optical density [OD], control: 100% vs. rhSCF: 37%). Almost identical results were obtained with HMC-1 cells (OD, control: 100% vs. rhSCF: 40 to 45%). As assessed by flow cytometry and monoclonal antibodies (mAbs) to c-kit, the SCF-induced decrease of c-kit mRNA in HMC-1 was associated with a substantial decrease in surface expression of c-kit (MFI, control: 100 +/- 21%, vs. MFI in cells incubated with rhSCF [100 ng/mL at 37 degrees C for 12 hours]: 8 +/- 2%, vs. MFI in cells incubated with rhSCF, 100 ng/mL, at 4 degrees C: 34 +/- 3%). The effects of rhSCF on c-kit expression in HMC-1 cells were dose- and time-dependent with maximum effects observed with 10-100 ng/mL of rhSCF after 4 to 12 hours. The SCF-dependent loss of c-kit was also accompanied by a decreased chemotactic response to rhSCF (control: 100%; rhSCF: 71 +/- 2%). This study shows that exposure of human lung MC and HMC-1 cells to recombinant SCF results in downregulation of c-kit mRNA and surface c-kit expression. These data may explain the partial loss of c-kit on MCs in areas of SCF overexpression.     

The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.     

Expression and functional activity of the IL-8 receptor type CXCR1 and CXCR2 on human mast cells

To further elucidate mechanisms involved in mast cell accumulation at sites of cutaneous inflammation, we have studied the ability of human leukemic mast cells (HMC-1 cells) to express functionally active IL-8 receptors. Expression of mRNA for both types of IL-8 receptors (CXCR1 and CXCR2) was demonstrated by PCR and of both proteins by flow cytometry. Binding and competition studies with 125I-labeled IL-8 and its homologue melanoma growth stimulating activity (125I-labeled MGSA) revealed two specific binding sites for IL-8, K1 = 1.1 x 10(11) M(-1) and K2 = 5 x 10(7) M(-1); and for MGSA, K1 = 2.8 x 10(10) M(-1) and K2 = 5 x 10(7) M(-1). This finding was supported by a dose-dependent rise of cytosolic free calcium concentration ([Ca2+]i) induced by both chemokines and to a lesser extent by the homologue neutrophil-activating peptide-2 (NAP-2). A significant migratory response of human leukemic mast cells (HMC-1) was observed with all three chemokines at a range from 10(-8) M to 10(-9) M. Moreover, the formation of cellular F-actin was induced in a rapid, dose-dependent fashion, with a maximally 1.7-fold increase at 10(-7) M. Using postembedding immunoelectron microscopy, we could show the expression of CXCRI on the cytoplasmatic membrane of isolated human skin mast cells whereas CXCR2 was located in mast cell-specific granules. These findings demonstrate for the first time the functional expression of both types of IL-8 receptors on human mast cells, suggesting a role for their ligands during mast cell activation and recruitment.     

Antigen-stimulated IL-4, IL-13 and IFN-gamma production by human T cells at a single-cell level

To understand the intricate balance and the coordinate expression of the Th1 and Th2 cytokines following a natural mode of T cell triggering, antigen-stimulated IL-4, IL-13 and IFN-gamma production was studied in primary peripheral blood mononuclear cell cultures at a single-cell level. Cells from filariasis patients who respond to parasite antigen by producing not only IFN-gamma but also IL-4 and IL-13 were stimulated with Brugia malayi adult worm antigen and analyzed for co-expression of cytokines by intracellular staining. IL-4 and IL-13 were frequently co-expressed (54% of IL-4+ cells stained for IL-13 and 29% of IL-13+ cells expressed IL-4 at all time points), whereas IFN-gamma expression was totally segregated from both IL-4 and IL-13. These data indicate that in human peripheral T cells the co-expression of the dominant Th1 and Th2 cytokines within a single cell is a rare event and that IL-13 is clearly more frequently associated with a Th2 than a Th1 type response in primary T cell cultures.     

IgE-induced passive sensitization of human isolated bronchi and lung mast cells

Passive sensitization of human isolated lung with serum from atopic asthmatic patients provides an opportunity to study the link between airway hyper-responsiveness and the allergic process. To directly demonstrate the role of immunoglobulin E (IgE) in the effect of the atopic serum, we have compared the effect of passively sensitizing both human bronchi and isolated lung mast cells with either serum from atopic asthmatic patients or human monoclonal IgE. Peripheral bronchi ( < 5 mm in internal diameter) were dissected out from human lung obtained at thoractomy and isometric contraction was studied in response to a variety of immunological stimuli according to the sensitization protocol. Mast cells were also isolated from human lung and histamine release was measured under similar experimental conditions. A contractile response was elicited by either the specific antigen or anti-IgE (0.6-600 ng.mL-1) but not anti-immunoglobulin G (IgG) 0.2-20 micrograms.mL-1) in airways sensitized with atopic serum (total IgE concentration of approximately 1,000 international units (IU).mL-1). The maximal contractile response to anti-IgE was 75 +/- 22% of the response to 1 mM acetylcholine. Similarly, anti-IgE released histamine from isolated lung mast cells sensitized with atopic serum up to 22.4 +/- 2% of total histamine measured within mast cells. When isolated airways or mast cells were sensitized with human monoclonal IgE (1,000 IU.mL-1), response to anti-IgE in terms of contractile response or histamine release, respectively, were not significantly different from those obtained following passive sensitization with atopic serum. Finally, the bronchial contractile response to anti-IgE depended not only on the concentration of anti-IgE but also on that of IgE (300-2,000 IU.mL-1) used to sensitize the airways. These results indicate that the effect of antigen or anti-IgE in peripheral bronchi passively sensitized with atopic serum is mimicked when sensitization is carried out directly with human monoclonal IgE.     

PDGF-alpha receptor subunit expression down-regulated by IL-1beta in human periodontal ligament cells

The responses of cells to the distinct PDGF isoforms have been correlated directly to the relative numbers of specific PDGF receptor subunits on the cell surface. The modulation of PDGF-alpha receptor subunits, the major subunit expressed in human periodontal ligament (PDL) cells, by cytokines present in the periodontal wound site, such as interleukin-1 (IL-1), may be an important factor influencing regenerative outcomes. The purpose of the present study was to examine the effects of IL-1 beta on PDGF-alpha receptor subunit expression in human PDL cells. Primary cultures of human PDL cells were treated with IL-1 beta over a range of concentrations. We assessed PDGF-alpha receptor subunits by examining the mitogenic responses of cells to PDGF-AA, specific binding of 125I-labeled PDGF-AA, immunofluorescent analysis of PDGF-alpha receptor subunits, and PDGF-alpha receptor subunit mRNA levels using Northern blot analysis. The results demonstrate a significant concentration-dependent decrease in 3H-thymidine incorporation in response to PDGF-AA following IL-1 beta treatment (p < 0.001). This decreased response correlated directly with IL-1-induced decreases in 125I-labeled PDGF-AA binding (p < 0.01), the numbers of immunolabeled PDGF-alpha receptor subunits, and in PDGF-alpha receptor subunit mRNA levels. However, when combined with TGF-beta, IL-1 beta did not show additional down-regulation in proliferative response to PDGF-AA or PDGF-alpha receptor subunits beyond that achieved with these factors individually. These experiments identify IL-1 beta, along with TGF-beta, as significant inhibitors of PDGF stimulation in human PDL cells, acting through the down-regulation of PDGF-alpha receptor subunit expression.     

IL-6 functions in cynomolgus monkeys blocked by a humanized antibody to human IL-6 receptor

A humanized antibody to the human interleukin-6 receptor (IL-6R), hPM-1, blocked the interleukin-6 (IL-6) functions in normal cynomolgus monkey lymphocytes in vitro. The binding activity of hPM-1 to non-human primate IL-6R was examined in peripheral blood lymphocytes by flow cytometry. PM-1 recognized the IL-6R on T lymphocytes of cynomolgus and rhesus monkeys, but did not on those of marmosets. The homology between human IL-6R and its cynomolgus monkey counterpart was 97.3% in the extracellular domain of the amino acid sequence, as determined by DNA sequencing of the PCR product from peripheral blood mononuclear cells. PM-1 inhibited two functional parameters in vitro in cynomolgus monkeys: (1), T-cell proliferation stimulated by phytohemaglutinin and human IL-6; (2), Immunoglobulin G-production evoked by Staphylococcus aureus Cowan-1- and human IL-6-stimulated B lymphocytes. These data show that hPM-1 binds to and functionally blocks the cynomolgus monkey IL-6 receptors.     

Regulation of the activity of secreted human lung mast cell tryptase by mast cell proteoglycans

When mast cells from human lungs were stimulated in vitro to degranulate, all of the tryptase secreted was found to be complexed with proteoglycans, three quarters with heparin proteoglycans and one quarter with chondroitin sulphate proteoglycans. Isolation of the tryptase-proteoglycan complexes by fibronectin affinity chromatography and gel filtration on a Sephacryl S-200 column gave the complexes an apparent Mr of 200000, suggesting the presence of heparin and chondroitin sulphate proteoglycans (Mrs=60000) and tryptase (Mr=134000) in a molar ratio of 1:1, equivalent to a mass ratio of about 0.45:1. However, analysis of the total mast cell releasate showed that it contained more proteoglycans (mass ratio of about 2:1) than was needed to complex tryptase. We could demonstrate that the releasate contained two proteoglycan fractions, one complexed (20%) with tryptase and the other not (80%). Incubation of the isolated tryptase-proteoglycan complexes led to rapid monomerisation and inactivation of tryptase, whereas the releasate, containing both complexed and free proteoglycans, retained its tryptase activity for up to at least 18 h. The results indicate that the majority of the proteoglycans secreted by stimulated lung mast cells, although not complexed with the secreted tryptase, are critical for the preservation of its activity.     

Cloning of the human IL-13R alpha1 chain and reconstitution with the IL4R alpha of a functional IL-4/IL-13 receptor complex

To study the anti-inflammatory mechanisms of inhaled corticosteroids and beta-agonist therapies, we evaluated basal and stimulus-induced superoxide production by human airway inflammatory cells obtained by bronchoalveolar lavage from normal volunteers before and after 3 weeks of an inhaled corticosteroid (flunisolide) and beta-agonist (metaproterenol). Assay of superoxide production by the bronchoalveolar lavage cells was performed in the presence of media alone or media containing phorbol ester by optical density determination of reduced ferricytochrome c at 550 nm. Interleukin-1 beta released from unstimulated cells and cells stimulated with lipopolysaccharide was quantitated by enzyme immunoassay. Interestingly, phorbol ester-stimulated superoxide production was strikingly inhibited (P < 0.05) by inhaled therapies, while stimulus induced Interleukin-1 beta production was not significantly affected (P = 0.12). Suppression of oxidant production by airway inflammatory cells may be a major mechanism for the beneficial anti-inflammatory effects of inhaled corticosteroids and beta-agonists.     

Differential regulation of IL-4 and IL-13 secretion by human basophils: their relationship to histamine release in mixed leukocyte cultures

Human basophils are an important source of IL-4, a cytokine that is central to the pathogenesis of allergic inflammation. Recent reports have indicated that these cells also generate IL-13, which shares a number of biologic properties with IL-4. We found basophils to be the major source of IL-13 produced in mixed leukocyte cultures following 20-h activation with a variety of stimuli. While the magnitude of IL-4 protein generated correlated with the percent histamine secreted (r = 0.8; p = 0.007), there was no relationship between the levels of IL-13 detected and the amount of either IL-4 or histamine in cultures activated with IL-3/anti-IgE. The induction of IL-13 secretion also occurred in response to IL-3 alone, without concomitant secretion of either IL-4 or histamine. Although previously shown to inhibit IL-4 secretion, the phorbol ester PMA was a potent stimulus for IL-13 generation from basophils, and this secretion was sensitive to the protein kinase C inhibitor, bisindolylmaleimide. In contrast, bisindolylmaleimide did not prevent cytokine secretion induced by either anti-IgE or IL-3. The immunosuppressant, FK506, while strikingly inhibiting the accumulation of IL-4 mRNA and the secretion of protein in response to IL-3/anti-IgE, had no effect on the generation of IL-13 in these cultures; the resistance was attributed to the IL-3-dependent signaling. Similarly, FK506 had no effect on the secretion of IL-13 in basophil cultures stimulated with PMA. This study suggests that multiple intracellular mechanisms control the generation of IL-13 in basophils, some of which are distinct from those regulating IL-4.     

Phagocytic and tumor necrosis factor alpha response of human mast cells following exposure to gram-negative and gram-positive bacteria

Recent studies have implicated rodent mast cells in the innate immune response to infectious bacteria. We report that cord blood-derived human mast cells (CBHMC) obtained from culture of cord blood progenitors phagocytozed and killed various gram-negative and gram-positive bacteria and simultaneously released considerable amounts of tumor necrosis factor alpha. Overall, the extent of the endocytic and exocytic response of CBHMC correlated with the number of adherent bacteria. Thus, human mast cells are intrinsically capable of mediating microbial recognition and of actively contributing to the host defense against bacteria.     

Interleukin (IL) 4 and IL-13 act on human lung fibroblasts. Implication in asthma

Airway hyperresponsiveness leading to subepithelial fibrosis is mediated by inflammatory cells activated by T helper (Th) 2-derived cytokines such as IL-4 and IL-5. By analyzing the phenotype and response of human lung fibroblasts derived from either fetal (ICIG7) or adult (CCL202) tissue as well as from a Th2-type stromal reaction (FPA) to IL-4 and IL-13, we provide evidence that human lung fibroblasts may behave as inflammatory cells upon activation by IL-4 and IL-13. We show that the three types of fibroblasts constitute different populations that display a distinct pattern in cell surface molecule expression and proinflammatory cytokine and chemokine release. All fibroblasts express functional but different IL-4/IL-13 receptors. Thus, while IL-4 receptor (R) alpha and IL-13Ralpha1 chains are present in all the cells, CCL202 and FPA fibroblasts coexpress the IL-13Ralpha2 and the IL-2Rgamma chain, respectively, suggesting the existence of a heterotrimeric receptor (IL-4Ralpha/IL-13Ralpha/IL-2Rgamma) able to bind IL-4 and IL-13. Stimulation with IL-4 or IL-13 triggers in the fibroblasts a differential signal transduction and upregulation in the expression of beta1 integrin and vascular cell adhesion molecule 1 and in the production of IL-6 and monocyte chemoattractant protein 1, two inflammatory cytokines important in the pathogenesis of allergic inflammation. Our results suggest that when activated by IL-4 and IL-13, different subsets of lung fibroblasts may act as effector cells not only in the pathogenesis of asthma but also in lung remodeling processes. They may also differentially contribute to trigger and maintain the recruitment, homing, and activation of inflammatory cells.     

Production of IL-1 alpha and IL-1 beta by human skin equivalents parasitized by Sarcoptes scabiei

Human skin equivalents (HSEs) were used as a model to investigate interleukin (IL)-1 alpha and IL-1 beta secretions by keratinocytes stimulated by Sarcoptes scabiei (SS). SS mites burrowed into the stratum corneum when placed on the surface of cultured HSEs. Mites lived for 14 days. Mites and mite products induced cells in the HSEs to secrete IL-1 alpha and IL-1 beta within 16 hr. Scabies mites induced production of greater amounts of IL-1 alpha than IL-1 beta. Hepatocyte growth factor in the culture medium at 3 and 30 ng/ml upregulated the secretions of both IL-1 alpha and IL-1 beta by mite-infested skin equivalents, whereas 10 ng/ml of IL-6 upregulated production of only IL-1 beta. Therefore, these cytokines were important immunomodulating factors influencing keratinocyte secretion of IL-1 alpha and IL-1 beta in vitro. The results of this study provide the first evidence that keratinocytes (possibly fibroblasts) in the skin produce these cytokines in response to scabies mites or other ectoparasitic arthropods. Because IL-1 alpha and IL-1 beta are potent inducers of inflammation and keratinocytes are among the first effector cells to encounter scabies mites and their products, these cells may be key initiators of the inflammatory/immune reaction to scabies.     

IL-10 synergizes with IL-4 and IL-13 in inhibiting lysosomal enzyme secretion by human monocytes and lamina propria mononuclear cells from patients with inflammatory bowel disease

Tissue injury and inflammation in inflammatory bowel disease (IBD) are associated with enhanced monocytic lysosomal enzyme release. In this study, peripheral monocytes and lamina propria mononuclear cells (LPMNC) were isolated from IBD patients and normal controls. Cells were stimulated with lipopolysaccharide after treatment with IL-13, IL-4, and IL-10, and enzyme secretion was assessed by using the corresponding p-nitrophenyl glycosides as substrates. Molecular forms of cathepsin D were examined to describe the mode of enzyme release. IL-10 and IL-4 strongly down-regulate enzyme secretion in IBD monocytes. IBD monocytes showed a diminished responsiveness to the inhibitory effect of IL-13. Impaired monocyte response was not found with combinations of IL-13 and IL-10 or IL-4 and IL-10. LPMNC from involved IBD mucosa showed significantly higher enzyme secretion compared with LPMNC from noninvolved IBD mucosa but responded inefficiently to either IL-4, IL-13, or IL-10 alone. However, combined treatment with IL-10 and IL-4 or IL-10 and IL-13 strongly suppressed enzyme release by these cells. Both the precursor and mature forms of cathepsin D were elevated in IBD patients. While IL-13 reduced mainly the precursor form, the effect of IL-4 and IL-10 concerns both the precursor and mature form of cathepsin D. Our results favor the potent clinical utility of combined treatment, thus improving chances of developing effective treatments for human IBD.     

Antitumor response of genetically engineered IL-2 expression to human esophageal carcinoma cells in mature T cell-defective condition

We examined whether antitumor effect could be produced by retrovirally expressed human interleukin-2 (hIL-2) gene in human esophageal cancer cells (T.Tn) using immunocompromised nude mice. Loss of tumorigenicity of hIL-2-producing T.Tn (T.Tn/hIL-2) cells inoculated subcutaneously was observed in contrast to continuous tumor growth of wild-type cells, although in vitro proliferation of T.Tn/hIL-2 cells remained the same as that of wild-type cells. The antitumor effect was also evidenced by the injection of T.Tn/hIL-2 cells into established tumors of wild-type cells. The injection significantly retarded the subsequent growth of wild-type tumors. Histological examination of regressing T.Tn/hIL-2 cells revealed necrotic areas and infiltration of several types of inflammatory cells. Treatment of nude mice with anti-asialoGM1 antibody did not influence the IL-2-mediated tumor rejection. Vaccination of nude mice with irradiated T.Tn/hIL-2 cells whose secretion of hIL-2 in amount was comparable to that of unirradiated cells did not develop protective immunity. Taken together, the antitumor effect achieved in nude mice by the inoculation of T.Tn/hIL-2 cells is mediated by non-T non-natural killer cells.     

Clinical correlates of bombesin-like peptide receptor subtype expression in human lung cancer cells

The importance of the expression of the autocrine growth system for bombesin-like peptides (BLPS) to the biological behavior of human lung cancer has not been determined. Three BLP receptor subtypes have been identified in human lung and lung cancer cells: gastrin-releasing peptide (GRP) receptor, neuromedin B (NMB) receptor, and bombesin receptor subtype 3 (BRS-3). The goals of this study were: (1) to determine BLP receptor subtype expression by human lung cancer cell lines by RT/PCR; (2) to evaluate possible clinical correlates of characteristics of the patients from whom the cell lines were derived with patterns of BLP receptor expression. Degenerate PCR primers were designed to amplify all known BLP receptors and yielded products from 19/20 small cell lung carcinoma (SCLC) and 12/13 non-small cell lung carcinoma (NSCLC) cell lines. GRP receptor was the most commonly expressed BLP receptor subtype, being detected in 17/20 SCLC and 11/13 NSCLC. Eleven of 20 SCLC expressed NMB receptors, and 5/20 expressed BRS-3, compared with 4/13 and 1/13, respectively, in NSCLC cell lines. Evaluation of the clinical data of the patients from whom the cell lines were derived revealed expected age, sex, smoking history and survival based on histology and stage. Patients from whom cell lines expressed GRP receptor experienced a better survival than those whose cell lines did not (367 +/- 274 days vs. 211 +/- 114 days), but the results were not statistically significant. RT/PCR analysis is a feasible, sensitive and specific means of determining BLP receptor expression in lung cancer cells and may yield prognostic information in patient tissue.