GM-CSF mRNA Expression Research Articles

Spatial expression of chemokines and cytokines mRNA in the largest preovulatory follicle of chicken

In the present experiment, we studied the spatial expression profiles of chemokines and cytokines mRNA in the granulosa (F1Gr) and theca (F1Th) layers of the largest preovulatory follicle in chicken using semi-quantitative PCR. The mRNAs of IL-1beta, IL-6, GM-CSF, chCXCLi2, chCCLi2, chCCLi4, chCCLi7, IFN-gamma, IL-4, IL-13, IL-10 and TGF-beta2 were expressed in the granulosa (F1Gr) and theca (F1Th) layers of the largest preovulatory follicle. However, the transcripts of IL-2 were not detected in any of the samples tested. Significantly higher levels of IL-6 and GM-CSF mRNA expression were noticed in F1Gr when compared to F1Th layer. Expression of chCXCLi2, a CXC chemokine, was almost similar in F1Gr and F1Th layers. However, the expression of CCL chemokines i.e. chCCLi2, chCCLi4, chCCLi7 mRNAs were almost 2 folds higher in F1Th layer in comparison to F1Gr layer. The expression of Th2 cytokines (IL-4 and IL-13) mRNA was noticed in F1Gr and F1Th layers with higher levels in the former. Expression of IFN-gamma mRNA was noticed in F1Gr and F1Th layers. Significantly higher level of TGF-beta2 expression was observed in F1Th in comparison to F1Gr layer. It was concluded from the present study that the mRNA expression of cytokines and chemokines are differentially regulated in the granulosa and theca layers of the largest preovulatory follicle in chicken.

Stimulation of Langerhans cells with ketoprofen plus UVA in murine photocontact dermatitis to ketoprofen

Ketoprofen (KP) clinically evokes the allergic type of photocontact dermatitis when applied to the skin and irradiated with ultraviolet A (UVA). We have established a murine model of photocontact dermatitis to KP, which is a T cell-mediated delayed type hypersensitivity. To further explore the mechanism underlying this sensitivity, we investigated whether KP plus UVA activates the antigen-presenting ability of Langerhans cells (LCs). We analyzed the expression of surface molecules on LCs in the murine epidermis treated with KP plus UVA by immunohistochemistry and flow cytometry. Changes in the cytokine expression of epidermal cells from KP-phototreated skin were also examined by real-time PCR. LCs became larger after treatment with KP plus UVA. The number of LCs was significantly decreased 2-3 days after KP phototreatment and recovered on day 5. A flow cytometric analysis revealed that KP plus UVA increased the percentage of LCs that highly expressed MHC class II, CD86, CD80, CD54 and CD40, whereas neither KP nor UVA alone enhanced the expression. KP phototreatment augmented the expression of I-A and CD86 on LCs in KP and UVA dose-dependent manners. A real-time PCR analysis of KP-phototreated skin showed that the expression of mRNA for IL-1alpha and GM-CSF was immediately increased after treatment. A photosensitizing regimen of KP plus UVA activates LCs at least partly by stimulating keratinocytes to produce cytokines. Two strains of mice (BALB/c and AKR) differ in responsiveness to KP and the difference is not related to the activation of keratinocytes.

별불가사리 추출물의 면역세포 활성화 효과

In this experiment, the effects of Asterina pectinifera extracts on the activation of immune cells were studied. An immune cell activating factor was partially purified from starfish, Asterina pectinifera, by means of physiological saline extraction, acetone precipitation and heating inactivation. Starfish extracts increased the proliferation of spleen cells and induced the production of IL-6 and IFN-γ by spleen cells. Also, it increased the proliferation of purified B cells and production of IgM and IgG in the presence of Asterina pectinifera extracts. Starfish extract self-induced NO synthesis in mouse macrophage cell line (RAW264.7). When cell lines was treated with extracts, the mRNA expression of inducible NO synthetase (iNOS), TNF-α, IL-6, and GM-CSF were markedly increased in RT-PCR analysis. Therefore starfish extract can self-activate spleen cells, B cells and macrophages. These results might be useful in further studies into a possible immune activating agent from the starfish, Asterina pectinifera, for the development of functional foods and drugs.

Clinical Observation on YiSuiShengXueGranule on Treating 156 Patients with .BETA.-Thalassemia Major and the Molecular Mechanism Study

To investigate the clinical effects and security of YiSuiShengXueGranule (YSSXG) on treating 156 patients with beta-thalassemia major. YSSXG was given orally to 156 patients with beta-thalassemia in GuangXi Autonomous Region (the high incidence area of beta-thalassemia in China) for 3 months as one therapeutic course, 3 times a day, 10 g each time (for children, the dose should be reduced properly according to their body weight and age), and no blood transfusion used during the course. Clinical symptoms and levels of hemoglobin (Hb), red blood cell (RBC), reticulocyte (Ret) and hemoglobin F (HbF) were observed before and after treatment, and side-effects were observed during the course. A 3-6 months follow up study was performed after withdrawal of YSSXG. And systemic gene analysis was conducted with PCR, SSCP-PCR, RT-PCR and DNA sequences analysis and mRNA differently expression technique, in order to study the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation. Levels of Hb, RBC, Ret and HbF obviously elevated, and clinical symptoms markedly ameliorated in patients after treated with YSSXG from the 1st to 3rd month (all p<0.01). Dynamical observation showed that the improvement of symptoms kept accordance with the elevation of hemorrheological indexes. The treatment was effective in 145 patients and ineffective in 11, and the total effective rate was 92.9%, without any adverse reaction founded. Follow-up studies showed the therapeutic effect could sustain for 3 to 4 months after drug-withdrawal. The molecular mechanism study showed: YSSXG did not change the genetic mutation type, but could obviously increase gamma/(beta+gamma) globin ratio, both gamma-globin mRNA and GM-CSF mRNA expression were significantly enhanced so as to induce HbF synthesis increasing after treated with YSSXG. YSSXG had obvious effects in treating beta-thalassemia by unlocking gamma-gene, increasing the gamma-globin expression and enhancing HbF synthesis so as to compensate for the gene defect. This study has provided a new path for the treatment of beta-thalassemia with Traditional Chinese Medicine.

Human intestinal epithelial cell cytokine mRNA responses mediated by NF-κB are modulated by the motility and adhesion process of Vibrio cholerae

Vibro cholerae, the etiological agent of cholera, colonizes the small intestine, produces an enterotoxin and causes acute inflammatory response at intestinal epithelial surface; the signals for such induction are still unknown. We determined the mRNA expression of proinflammatory and anti-inflammatory cytokines in Int407 cells following infection with V. cholerae or its mutants by semi-quantitaive and quantitative real-time RT-PCR. V. cholerae induces the coordinated expression and up-regulation of IL-1α, IL-6, GM-CSF and MCP-1 and down-regulation of TGF-β in Int407 cells. While the pathogenecity of V. cholerae was found to be a possible determinant in modulation of IL-1α and TGF-β, both IL-6 and MCP-1 OmpU might modulate induction . Significant reduction in IL-1α, GM-CSF and MCP-1 mRNA expression was observed upon infection with the less motile and less adherent strain O395YN. This association is supported by the absence of nuclear translocation of NF-κB (p50 subunit) upon infection with O395YN in contrast to wild-type. Moreover, TPCK treatment prior to V. cholerae infection indicated that proinflammatory cytokine gene expression in Int407 cells is NF-κB mediated. Thus, V. cholerae induces proinflammatory cytokine response in Int407 cells, which is mediated by NF-κB and is modulated, in part, by adherence or motility of this organism.

Evaluation of biologic activity of tryptase secreted from blast cells in acute myeloid leukemia

A number of autocrine and paracrine growth regulators are considered to be involved in the survival and proliferation of blast cells in acute myeloid leukemia (AML). We have recently shown that blast cells in a group of patients with AML produce and secrete the mitogenic enzyme tryptase. In the present study, we examined functional effects of tryptase in the context of AML. As assessed by 3H-thymidine uptake experiments, tryptase-containing serum from patients with AML as well as heparin-complexed recombinant tryptase were found to promote the proliferation of cultured bone marrow- and lung fibroblasts in a dose-dependent manner. A neutralizing antibody against human β-tryptase was found to diminish these growth-stimulatory effects of serum-tryptase in all patients examined. Tryptase also induced the expression of mRNA for GM-CSF and SCF, two cytokines known to promote growth of AML cells, in cultured bone marrow fibroblasts. Neither recombinant tryptase nor tryptase-rich serum of AML patients, showed an effect on the growth of leukemic blast cells irrespective of the FAB category or expression of protease-activated receptor (PAR)-2, a putative molecular target of tryptase. Together, tryptase is secreted from AML blasts as a biologically active molecule that may exhibit paracrine rather than autocrine effects in AML.

Interleukin-17 augments tumor necrosis factor-α-induced granulocyte and granulocyte/macrophage colony-stimulating factor release from human colonic myofibroblasts

Interleukin (IL)-17 is a newly identified T-cell-specific cytokine. In this study, we investigated the effects of IL-17 on colony-stimulating factor (CSF) release in human colonic subepithelial myofibroblasts (SEMFs). CSF release and mRNA expression were determined by enzyme-linked immunosorbent assay (ELISA) and Northern blotting, respectively. Nuclear factor (NF)-kappaB- and activating protein (AP-1)-DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSAs). Unstimulated cells secreted a small amount of granulocyte G- and granulocyte/macrophage (GM)-CSF, and a considerable amount of M-CSF. IL-17 weakly enhanced G-CSF release, but did not affect GM- and M-CSF release. IL-17 selectively enhanced tumor necrosis factor (TNF)-alpha-induced G- and GM-CSF release. The combination of IL-17 plus TNF-alpha induced a marked increase in NF-kappaB- and AP-1-DNA binding activities. The adenovirus-mediated transfer of a stable form of IkappaBalpha and/or a dominant negative mutant of c-Jun markedly inhibited the IL-17 plus TNF-alpha-induced G- and GM-CSF mRNA expression. Furthermore, a stability study showed that IL-17 plus TNF-alpha markedly enhanced the stability of G- and GM-CSF mRNA. IL-17 augments TNF-alpha-induced G- and GM-CSF release via transcriptional and posttranscriptional mechanisms.

Gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived mesenchymal stem cell

Mesenchymal stem cells (MSCs), which are adherent stromal cells of a nonhematopoietic origin, have the ability to give rise to various differentiated cell types. MSCs regulate localization, self-renewal and differentiation of hematopoietic stem cells (HSCs) due to MSCs' secretion of cytokines and growth factors, the cell-to-cell interactions and the influence of the extracellular matrix proteins. Using RT-PCR analysis, we examined the expression levels of cytokines and growth factors from MSCs and their differentiated cell types, including osteoblasts, adipocytes and endothelial cells. Cytokine and growth factor genes, including IL-6, IL-8, IL-11, IL-12, IL-14, IL-15, LIF, G-CSF, GM-CSF, M-SCF, FL and SCF, were found to be expressed in the MSCs. In contrast, there was no IL-1α, IL-1β, or IL-7 expression observed. The IL-12, IL-14, G-CSF, and GM-CSF mRNA expression levels either disappeared or decreased after the MSCs differentiated into osteoblasts, adipocytes, and endothelial cells. Among the differentiated cells derived from MSCs, osteoblasts, adipocytes, and endothelial cells expressed the osteopontin, aP 2, and the VEGFR-2 gene, respectively. These profiles could help determine future clinical applications of MSCs and their derivatives for cell therapy.

Effects of microcystin-LR on patterns of iNOS and cytokine mRNA expression in macrophagesin vitro

The presence of cyanobacterial toxins in drinking and recreational waters represent a potential health hazard to the public. Microcystin-LR (MC-LR) is the most commonly encountered toxin and is a potent cyclic heptapeptide hepatotoxin produced by cyanobacteria. In this study, the immunomodulation by MC-LR of BALB/c mice peritoneal macrophages was investigated in vitro on mRNA levels of induced nitric oxide synthase and multiple cytokines by reverse-transcription polymerase chain reaction (RT-PCR). Lavaged peritoneal macrophages were incubated for 6 h with lipopolysaccharide (LPS) at a concentration of 100 microg/L and MC-LR at doses of 1, 10, 100, and 1000 nmol/L. Total RNA was extracted from the incubated macrophages, and then the levels of mRNA for induced nitric oxide synthase (iNOS), IL-1beta, TNF-alpha, GM-CSF, and IFN-gamma were detected. The results showed that expression of mRNA for iNOS, IL-1beta, TNF-alpha, GM-CSF, and IFN-gamma decreased significantly compared to the positive control (LPS only). These results have led us to propose the need for the establishment of a survey of the immunotoxicity of microcystins.

Modulation of Proinflammatory Responses to Pneumocystis carinii f. sp. muris in Neonatal Mice by Granulocyte-Macrophage Colony-Stimulating Factor and IL-4: Role of APCs

Clearance of Pneumocystis carinii f. sp. muris (PC) organisms from the lungs of neonatal mice is delayed due to failure of initiation of inflammation over the first 3 wk after infection. The ability of neonatal lung CD11c(+) dendritic cells (DCs) to induce Ag-specific T cell proliferative responses was significantly reduced compared with adult lung DCs. However, neonatal bone marrow-derived DCs were as competent at presenting PC Ag as were adult bone marrow-derived DCs. Because GM-CSF mRNA expression and activity were significantly reduced in neonatal lungs compared with adults, we treated neonates with exogenous GM-CSF and IL-4 and found enhanced clearance of PC compared with untreated neonates. This was associated with increased lung TNF-alpha, IL-12p35, and IL-18 mRNA expression, indicating enhanced innate immune responses. Cytokine-treated mice had marked expansion of CD11c(+) DCs with up-regulated MHC-II in the lungs. Moreover, increased numbers of activated CD4(+)CD44(high)CD62L(low) cells in the lungs and draining lymph nodes suggested improved Ag presentation by the APCs. Together these data indicate that neonatal lungs lack maturation factors for efficient cellular functioning, including APC maturation.

Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation

The aim of this study was to investigate the stimulating effect of low-dose radiation (LDR) on bone marrow hematopoietic progenitor cell (HPC) proliferation and peripheral blood mobilization. Mice were exposed to 25- to 100-mGy x-rays. Bone marrow and peripheral blood HPCs (BFU-E, CFU-GM, and c-kit+ cells) were measured, and GM-CSF, G-CSF, and IL-3 protein and mRNA expression were detected using ELISA, slot blot hybridization, and Northern blot methods. To functionally evaluate LDR-stimulated and -mobilized HPCs, repopulation of peripheral blood cells in lethally irradiated recipients after transplantation of LDR-treated donor HPCs was examined by WBC counts, animal survival, and colony-forming units in the recipient spleens (CFUs-S). 75-mGy x-rays induced a maximal stimulation for bone marrow HPC proliferation (CFU-GM and BFU-E formation) 48 hours postirradiation, along with a significant increase in HPC mobilization into peripheral blood 48 to 72 hours postradiation, as shown by increases in CFU-GM formation and proportion of c-kit+ cells in the peripheral mononuclear cells. 75-mGy x-rays also maximally induced increases in G-CSF and GM-CSF mRNA expression in splenocytes and levels of serum GM-CSF. To define the critical role of these hematopoietic-stimulating factors in HPC peripheral mobilization, direct administration of G-CSF at a dose of 300 microg/kg/day or 150 microg/kg/day was applied and found to significantly stimulate GM-CFU formation and increase c-kit+ cells in the peripheral mononuclear cells. More importantly, 75-mGy x-rays plus 150 microg/kg/day G-CSF (LDR/150-G-CSF) produced a similar effect to that of 300 microg/kg/day G-CSF alone. Furthermore, the capability of LDR-mobilized donor HPCs to repopulate blood cells was confirmed in lethally irradiated recipient mice by counting peripheral WBC and CFUs-S. These results suggest that LDR induces hematopoietic hormesis, as demonstrated by HPC proliferation and peripheral mobilization, providing a potential approach to clinical application for HPC peripheral mobilization.

IL-17 and IL-17F modulate GM-CSF production by lung microvascular endothelial cells stimulated with IL-1β and/or TNF-α

In this study, we investigated the roles of CD4 T cell cytokines IL-17 and IL-17F in GM-CSF production from lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not induce GM-CSF release from LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1β- and TNF-α-induced GM-CSF mRNA expression and production, whereas IL-17F had an enhancing effect on IL-1β-induced GM-CSF production, but a marked inhibitory effect on TNF-α-induced secretion. GM-CSF production was further enhanced with the combination of three cytokines IL-1β, TNF-α and IL-17 or IL-17F. Additionally, when Th1 or Th2 cytokine was combined with IL-1β or TNF-α, both Th1 and Th2 cytokines had a modest stimulatory effect on TNF-α-induced GM-CSF production, whereas IL-4 and IFN-γ profoundly attenuated IL-1β-induced secretion. Moreover, the regulation by IL-17 plus Th1 or Th2 cytokine of GM-CSF production from LMVECs treated with IL-1β or TNF-α was dependent on the concentration of IL-17. Our findings indicate that IL-17 and IL-17F play a differential regulatory role in GM-CSF production by LMVECs stimulated with IL-1β and/or TNF-α, which is sensitive to Th1 and Th2 cytokine modulation.

T Cell Epitope-Specific Defects in the Immune Response to Cat Allergen in Patients with Atopic Dermatitis

Atopic dermatitis (AD) is often associated with high titer IgE antibodies (ab) to allergens, and IL-10-mediated regulation of IFN-gamma has been proposed to contribute to this IgE ab production. However, the relevance of IL-10 and IFN-gamma to IgE associated with AD has not been examined in the context of an allergen-specific system. Analysis of PBMC responses in vitro showed deficient T cell proliferation to overlapping IL-10- (peptide (P) 2:1) and IFN-gamma- (P2:2) inducing chain 2 major epitopes of cat allergen (Fel d 1) in cultures from sensitized AD patients (mean IgE to cat=20.9 IU/ml). Diminished IFN-gamma induction by Fel d 1 and P2:2, along with elevated peptide-induced IL-10 (except for P2:1) was observed in PBMC cultures from AD subjects compared with non-AD (sensitized and non-sensitized) subjects. Neither T cell proliferation nor IFN-gamma production to chain 2 epitopes could be restored by anti-IL-10 mAb in cultures from sensitized AD subjects. Moreover, allergen avoidance was associated with a paradoxical decrease in both IL-10 and IFN-gamma in peptide-stimulated PBMC from these subjects. Control of IFN-gamma production to chain 2 epitopes by IL-10 may be relevant to sensitization status. Development of high titer IgE ab in AD could reflect a failure of this mechanism.

Differential effects of fluticasone propionate (FP) on gene expression in airway epithelial cells

Rationale Airway epithelial cells play roles in both innate immunity and inflammatory responses in the airways. To determine whether glucocorticoids regulate airway epithelial responses uniformly, we investigated the effect of the potent glucocorticoid FP on expression of both innate immune effectors and inflammatory mediators after stimulation with the Toll-like receptor 3 ligand double stranded RNA (dsRNA). Methods BEAS-2B airway epithelial cells were treated with FP (10 −7 M) for 2 hours before stimulation with dsRNA (25 μg/ml) for 18 hours. RNA was isolated for real-time RT-PCR analysis and cell supernatants were analyzed for protein by ELISA. Results In ≥3 experiments, stimulation of AEC with dsRNA increased mRNA levels as indicated: Complement factor B (mean, 28 fold), factor H (30 fold), SAA (78 fold), secretory leukocyte protease inhibitor (SLPI) (2.4 fold), RANTES (3028 fold), IL-8 (1025 fold), MIP-1α (101 fold), GM-CSF (1622 fold), IL-1β (34 fold). Treatment with FP did not inhibit induction of mRNA for factor B, factor H, SAA or SLPI. FP did inhibit expression of mRNA for IL-8, MIP-1α, RANTES, IL-1β and GM-CSF induced by dsRNA (in all cases ≥58% inhibition, n=3-5, p<0.05). Similar results were obtained in human primary bronchial epithelial cells (n=1). ELISA of SAA, GM-CSF and IL-8 confirmed the mRNA expression results. Conclusion These results suggest that glucocorticoids may spare innate immune responses in airway epithelial cells while they inhibit expression of inflammatory mediators.

15d-PGJ 2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-κB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2), a natural ligand for peroxisome proliferator-activated receptor γ, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [ 3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ 2 inhibited Ox-LDL-induced [ 3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ 2 or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ 2. The suppression of GM-CSF promoter activity by 15d-PGJ 2 and 2-cyclopenten-1-one was mediated through reduction of NF-κB binding to GM-CSF promoter. These results suggest that 15d-PGJ 2 inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-κB inactivation.

Clinical efficacy and molecular mechanism of nourishing shen and supplementing marrow principle in treating β-thalassemia

Objective: To explore the possibility of using traditional Chinese medicine (TCM) in treating β-thalassemia, its clinical effect and molecular mechanism of the action.Methods: According to the TCM theory of “Shen producing marrow”, the composite recipe, Yisui Shenxueling Granule (YSSXL), consisting of Chinese drugs for nourishing Shen and supplementing marrow (NS & SM) was given orally to 78 patients with β-thalassemia (49 of the severe type and 29 of moderate type), 3 times a day, 10 g each time (for children, the dose would be reduced properly), with 3 months as one therapeutic course, and no blood transfusion used in the course. The clinical therapeutic efficacy and hematologic parameters in patients were observed, and systemic gene analysis was conducted with PAGE, PCR, PCR-SSCP, RT-PCR and DNA sequences analysis and mRNA detection, in order to study the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation.Results: YSSXL showed obvious therapeutic effect in treating β-thalassemia. Gene analysis revealed that it did not change the genetic mutation type, but could obviously increase hemoglobin, fetal hemoglobin (HbF), γ/(β+γ) globin ratio, γ-globin mRNA expression and GM-CSF mRNA expression in patients, as well as the GM-CSFmRMA in marrow of mice after60Co radiation.Conclusion: YSSXL has a remarkable therapeutic effect on β-thalassemia, and its possible mechanism is its action in unlocking γ-gene, increasing the γ-globin expression and enhancing HbF synthesis so as to compensate for the gene defect. This study has opened a new path for the treatment of β-thalassemia with TCM.

Interaction between the immune system and tongue squamous cell carcinoma induced by 4-nitroquinoline N-oxide in mice

Squamous cell carcinoma of the oral cavity (SCC) accounts for 3% of cancers in the western world and 40% of cancers in India. The overall 5-year survival rate is only 50%. Most of the lesions appear intra-orally on the tongue. Results from a previous study demonstrated a significant increase in T and B-lymphocytes under the transformed epithelium when examining human lesions of hyperkeratosis, dysplasia and carcinoma of the tongue. In order to investigate the interaction between the host immunity and SCC, carcinogen induced SCC of the tongue was studied in mice. The water-soluble carcinogen, 4 nitroquinoline N-oxide (4NQO), was applied to BALB/c mice tongues and produced tongue SCC after a long incubation period of several months. Immunologic properties were examined systemically in the spleens and locally, at the tumor site. Examination of spleen lymphocytes from 4NQO induced mice revealed enlargement of the spleens and a significant decrease in the CD3, CD4, CD8 and CD19 cells. In the tongues, expression of TGF-β, TNF-α, GM-CSF, and IL-1β mRNA were detected. TNF-α protein was detected in the affected tongues using immunoassays. mRNA expression of TNF-α was detected in the cancerous epithelium when extracted from the connective tissue. CD11b and CD3 cells were detected in the connective tissue under the developing carcinoma. CD11b positive cells were more prominent. The infiltrate was very scattered and not prominent as the infiltrate in the human tongue tissues. These results indicate that the growing tumor affected the immune response around the tumor and systemically. Most of the cytokines, which appeared in the affected tongues, originated from the tumor surroundings, but TNF-α was found also in the tumor. The interaction between the tumor and immune response components is important for diagnosis and treatment purposes.

Vitamin D 3 and analogues modulate the expression of CSF-1 and its receptor in human dendritic cells

The active vitamin D 3-metabolite 1,25(OH) 2D 3 inhibits the interleukin 4/granulocyte–macrophage colony-stimulating factor (IL-4/GM-CSF)-induced differentiation of human monocytes into dendritic cells without altering survival. Colony-stimulating factor 1 (CSF-1) is an important survival factor for cells of the monocytic lineage. We therefore investigated whether the inhibitory activity of 1,25(OH) 2D 3 is paralleled by a regulation of CSF-1 and its receptor. Purified human monocytes were cultured together with IL-4/GM-CSF in the presence of 1,25(OH) 2D 3, its analogue tacalcitol, the low-affinity vitamin D receptor ligand 24,25(OH) 2D 3, or the solvent ethanol for up to 5 days. Expression of CSF-1, CSF-1R, and GM-CSF mRNA was measured by RT-PCR. Protein secretion for CSF-1 was measured by ELISA, expression of CSF-1R by flow cytometry. The results showed that 1,25(OH) 2D 3 and tacalcitol significantly up-regulated CSF-1 mRNA-expression and protein secretion in a dose-dependent manner. The effect of 1,25(OH) 2D 3 occurred already after 1 h of pre-treatment. In contrast, CSF-1R mRNA- and cell surface-expression was down-regulated simultaneously. The solvent ethanol and 24,25(OH) 2D 3 were without effect. GM-CSF mRNA expression was not modulated in 1,25(OH) 2D 3-treated cells. These data point towards a distinct and specific regulation of CSF-1 and its receptor by 1,25(OH) 2D 3 and its analogue tacalcitol in human monocytes which parallels the inhibition of differentiation into dendritic cells without altering survival.

Nuclear factor-kappa B activation in the nasal polyp epithelium: relationship to local cytokine gene expression.

A panel of cytokines has been found to be important for eosinophil accumulation and activation in nasal polyps. The aims of this study were to ascertain whether the activation of nuclear factor-kappa B (NF-kappaB) occurred in the polyp epithelium, and to examine the relationship between the degree of activation and local cytokine gene expression. Nasal polyp specimens were obtained from 26 untreated patients. The proportion of nuclear translocation of the NF-kappaB p50 subunit in the polyp epithelium was quantitatively analyzed by a combination of fluorescent immunohistochemistry and a laser scanning confocal microscope image system. The levels of GM-CSF, IL-5, IL-8, IL-16, and eotaxin mRNA expression in the same speci-mens were measured using reverse transcription-polymerase chain reaction. Both cytoplasmic and nuclear localization of the p50 subunit was observed mainly in the epithelial layer in all specimens. The percentages of epithelial cells with nuclear translocation ranged from 4.5% to 40.5% (median, 18%). Significant correlations were observed between the degree of epithelial NF-kappaB activation and the levels of IL-8, IL-16, and eotaxin mRNA expression (r = 0.468, 0.47, and 0.739, respectively). The activation of NF-kappaB in the nasal polyp epithelium is responsible for the recruitment of inflammatory cells, particularly eosinophils, through the initiation of the transcriptional pathway of the related cytokines. The increased NF-kappaB activity in the polyp epithelium may reflect hypersensitivity to unknown stimuli.

Cross-linking of human FcgammaRIIIb induces the production of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor by polymorphonuclear neutrophils.

We have reported that human autoantibodies reacting with the polymorphonuclear neutrophil (PMN)-anchored FcgammaRIIIb (CD16) protect these cells from spontaneous apoptosis. In this study, we used anti-CD16 F(ab')(2) to delineate the mechanism(s) whereby the PMN life span is extended. As documented using four methods, CD16 cross-linking impeded spontaneous apoptosis, whereas anti-CD18 F(ab')(2) exerted no effect. Incubation of PMNs with anti-CD16 prevented the up-regulation of beta(2) integrins, particularly CD11b, which is the alpha-chain of complement receptor type 3, but also CD18, which is its beta-chain, as well as CD11a and CD11c. Anti-CD16-conditioned supernatant of PMNs diminished the percentage of annexin V-binding fresh PMNs after another 18 h in culture, whereas the negative control anti-CD18 had no effect. The expression of mRNA for G-CSF and GM-CSF was induced by anti-CD16, followed by the release of G-CSF and GM-CSF in a dose-dependent manner. Anti-G-CSF and anti-GM-CSF mAbs abrogated the antiapoptotic effect of the related growth factors. The delay in apoptosis was accompanied by a down-regulated expression of Bax, and a partial reduction of caspase-3 activity. These data suggest an autocrine involvement of anti-CD16-induced survival factors in the rescue of PMNs from spontaneous apoptosis. Thus, apoptosis of aged PMNs can be modulated by signaling through FcgammaRIIIb, which may occur in patients with PMN-binding anti-FcgammaRIIIb autoantibodies.