IL-24 modulates IFN-gamma expression in patients with tuberculosis

IL-24 is a newly described member of the IL-10 family. We previously demonstrated that PBMC from TB patients exhibited low levels of IL-24 and IFN-gamma compared to subjects with latent tuberculosis infection (LTBI). In order to investigate the role of IL-24 in IFN-gamma expression in TB patients, we stimulated PBMC from individuals with LTBI or TB patients with the Mtb-specific antigen, early secretory antigenic target-6 (ESAT-6) and measured cytokine expression using quantitative real-time PCR (qPCR). Exogenous IL-24 increased IFN-gamma expression in PBMC obtained from TB patients while neutralization of IL-24 reduced IFN-gamma expression in PBMC from subjects with LTBI. Exogenous IL-24 enhanced IFN-gamma expression by increasing expression of IL-12 family cytokines, including IL-12alpha, IL-12beta, IL-23alpha and IL-27, and by reducing FOXP3 expression in PBMC from TB patients. This is the first demonstration that IL-24 may play an important role in IFN-gamma expression following infection with Mtb.     

Distinct patterns of gene expression in the skin lesions of atopic dermatitis and psoriasis: a gene microarray analysis

BACKGROUND: Atopic dermatitis (AD) and psoriasis are the two most common chronic inflammatory skin diseases. Both of these diseases have distinct clinical findings and specific inflammatory cell infiltrates. Previous reports have focused individually on one or two genes or gene products in the lesions of both skin diseases. However, they have not captured the complex gene expression that must occur to induce specific cellular infiltrates in the skin lesions of these two diseases. DNA microarray studies allow the simultaneous comparison of thousands of messenger RNAs that may identify the disease-specific pattern of tissue inflammatory responses. OBJECTIVE: To compare the complex gene expression pattern of AD versus psoriasis skin lesions. METHODS: RNA was extracted from skin biopsy specimens of 6 patients with AD and 7 patients with psoriasis and analyzed with the use of Hu-U95Av.GeneChip microarrays. To confirm GeneChip results, real-time PCR of selected genes were performed. RESULTS: In AD skin, a total of 18 genes including the CC chemokines, CCL-13/MCP-4, CCL-18/PARC, and CCL-27/CTACK showed a statistically significant, >2-fold increase of gene expression compared with psoriasis. In psoriasis skin, a total of 62 genes including CCL-4/MIP-1beta, CCL-20/MIP-3alpha, CXCL-2/GRO-beta CXCL-8/IL-8, and CXCR2/IL-8R showed a >2-fold increase of gene expression compared with AD skin. Real-time PCR confirmed several of these GeneChip results. CONCLUSIONS: These results show a very distinctive gene expression pattern in AD as compared with psoriasis that may explain several features of AD and psoriasis including the specific inflammatory cell infiltrates observed in these disorders, that is, T(H)2 cells, eosinophils, and mast cells in AD and T(H)1 cells and neutrophils in psoriasis. Such observations may contribute to a characteristic "signature" for these two skin diseases.     

Interferon-gamma production in atopic dermatitis: a role for prostaglandins?

Peripheral blood mononuclear cells (PBMC) from patients with atopic dermatitis (AD) have a reduced capacity to produce interferon-gamma (IFN-gamma) in vitro, in response to phytohaemagglutinin (PHA) when compared to healthy non-atopic controls. This defect appears to correlate closely with the severity of AD at the time of sampling, with less IFN-gamma being produced by cells from patients with more severe disease. Enhanced production of IFN-gamma was observed as the patients clinical symptoms improved. In addition, IFN-gamma production could be increased by either pre-culturing the cells for 3 days prior to PHA stimulation or by addition of indomethacin to the culture medium. These observations suggest that the mechanism of reduced IFN-gamma production in AD is unlikely to be due to an intrinsic cellular defect. The possibility that prostaglandins mediate the suppressed production of IFN-gamma in AD was supported by demonstrating that exogenous prostaglandin E2 (PGE2) inhibited IFN-gamma production in PHA-stimulated PBMC. PGE2 at a physiological concentration (10(-9) M) was also shown to enhance interleukin 4 induction of IgE synthesis by PBMC cultures. Our data suggest that alterations in prostaglandin metabolism play a crucial role in the pathogenesis of AD by inhibiting the production of IFN-gamma.     

IL-9 is associated with an impaired Th1 immune response in patients with tuberculosis

Although a defective Th1 response has been demonstrated in patients infected with Mycobacterium tuberculosis (Mtb), the mechanisms leading to this defect are not well understood. To study the immune response to Mtb infection, we stimulated PBMC from individuals with latent tuberculosis infection (LTBI) or patients with tuberculosis (TB) with the Mtb specific antigen early secretory antigenic target-6 (ESAT-6). mRNAs for a panel of cytokines were measured using quantitative real-time PCR (qPCR). PBMC from TB patients exhibited low levels of IFN-gamma, IL-12alpha, IL-12beta, and IL-23 mRNA but high levels of IL-9 mRNA. Sera from TB patients blocked the differentiation and function of dendritic cells from TST negative (TST-) donors. Exogenous IL-9 reduced IFN-gamma mRNA expression in PBMC from LTBI by 30% (n=4) and neutralization of IL-9 restored the IFN-gamma mRNA expression in PBMC from TB patients by 66% (n=8). Thus, increased expression of IL-9 may contribute to the development of TB.     

Intracellular interferon-gamma (IFN-gamma) production in normal children and children with atopic dermatitis

A reduction in the in vitro production of IFN-gamma has been consistently described in atopic dermatitis (AD). Whether this reduction is due to a decrease in the population of peripheral blood mononuclear cells (PBMC) producing IFN-gamma or reduced IFN-gamma production per cell, or a combination of both is not clear. We have examined the intracellular production of IFN-gamma in children with AD and in healthy non-atopic controls. As Staphylococcus aureus colonization is a feature of childhood AD, and is postulated to contribute to the cutaneous inflammation in atopic dermatitis, S. aureus and Staphylococcal enterotoxin B (SEB) were used to activate PBMC. Stimulated PBMC from subjects with AD had significantly fewer IFN-gamma-containing cells in response to SEB (P < 0.001) and S. aureus (P < 0.01) than normal non-atopic children. In addition, SEB-stimulated PBMC from children with AD had less IFN-gamma per cell than normal non-atopic children (P < 0.01). Reduction in the proportion of cells containing IFN-gamma was seen in CD4+, CD8+ and natural killer (NK) cells in PBMC from children with AD. Our findings indicate that reduced production of IFN-gamma observed in childhood AD is due to both a decrease in the number of IFN-gamma-producing cells and a reduced amount of IFN-gamma production per cell. Furthermore, we found that this defect was not confined to CD4+ T cells, suggesting a more generalized defect in IFN-gamma production in childhood AD.     

Evidence that defective interferon-gamma production in atopic dermatitis patients is due to intrinsic abnormalities.

The in vitro production of interferon-gamma (IFN-gamma) in 19 atopic dermatitis (AD) patients was compared with that of 12 controls. IFN-gamma production by phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) was profoundly diminished in AD patients, whereas the proliferative response was similar to that of control PBMC. The addition of 40 U/ml of interleukin-2 (IL-2) to the cultures failed to restore IFN-gamma production. Similarly, removal of adherent cells also had no effect. Reduced IFN-gamma secretion was observed after stimulation with the CD3 monoclonal antibody OKT3, ionomycin + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or with high levels of IL-2 (200 U/ml). There were increased proportions of CD4+ T helper/inducer cells and decreased proportions of CD8+ T cytotoxic-/suppressor cells and CD16+ natural killer (NK) cells in AD patients. This resulted in an increased CD4/CD8 ratio as compared with controls, but no correlation was observed between numbers of T cell subpopulations and IFN-gamma generation. However, a significant correlation was found between IFN-gamma generation in vitro and IgE serum concentration in AD patients. The data suggest that the decreased production of IFN-gamma by AD patients is due to intrinsic differences in capacity to produce this cytokine and is not the result of differences in regulatory cell interactions. Moreover, the findings indicate that decreased production of IFN-gamma may be an important factor in the pathogenesis of this disease.     

Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis by differential display

To identify the genes related to atopic dermatitis (AD), we compared gene expression in eosinophils from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils. Gene expression was monitored by fluorescent differential display (DD) and real-time RT-PCR. Eighteen new sequences, including expressed sequence tags (ESTs), were expressed at higher levels in eosinophils from AD patients than in those from healthy volunteers. The functions of most of these genes are unknown. We found no correlation between the expression of a particular gene and clinical markers such as the number of eosinophils and the amount of IgE. Multivariate analysis of the gene expression data in each sample showed a very high coefficient of correlation among the copy numbers of each gene. The genes under investigation were also expressed in cultured blood eosinophils after IL-4, IL-5 and IFN-gamma stimulation. We were able to predict the function of some of the sequences by scanning for homologies within either the human or mouse genome databases. The mouse counterpart of one of these genes, intersectin 2, was expressed dramatically, as measured by ear edema, in 1-fluoro-2,4-dinitrobenzene-induced mouse contact dermatitis and in NC/Nga mouse dermatitis.     

In vitro responsiveness to IL-18 in combination with IL-12 or IL-2 by PBMC from patients with bronchial asthma and atopic dermatitis

Interleukin (IL)-18 is a proinflammatory cytokine and is now recognized as an important regulator of both helper T cells (Th) 1 and 2 cytokine production. An increased IL-18 secretion has been reported in patients with allergic disorders. It is predominantly produced by activated macrophages, and synergizes with IL-12 and IL-2 to induce IFN-gamma synthesis, thereby promoting Th1 cytokine response. Paradoxically, IL-18, by itself, strongly induces immunoglobulin (Ig) E and allergic inflammation, indicating a role for IL-18 in promoting Th2 response. We investigated the inducing effect in vitro of combining IL-18 and Il-12 or Il-2 on Th1- and Th2-type cytokines production by peripheral blood mononuclear cells (PBMC) from patients with allergic diseases. PBMC derived from 44 allergic patients [23 bronchial asthma (BA) and 21 atopic dermatitis (AD)] and 20 healthy controls were cultured with IL-18 in the presence of phytohemagglutinin (PHA) and IL-12 or IL-2. The levels of IFN-gamma, IL-13, and IL-4 in the culture supernatants were measured using enzymatic immunoassaying. IFN-gamma production was detected in all cultures from nonallergic controls stimulated with IL-18 in the presence of IL-12; however, the results for five BA patients and five AD patients were under the detection limit for IFN-gamma. In collaboration with IL-2, IL-18 was able to induce IFN-gamma production by PBMCs from all nonallergic controls and all allergic patients, with the exception of one AD patient. Synergistic induction of IL-13 production was found in cultures with IL-18 + IL-2, and the IL-13 induction was significantly increased in BA patients when compared with that in nonallergic controls (P = 0.006). The stimulation by IL-18, even in combination with IL-2, failed to induce IL-4 production by PBMC from both nonallergic controls and allergic patients. Although the induction of IFN-gamma by IL-18 + IL-12 was impaired in around a quarter of the allergic patients, the impairment of the IFN-gamma production was completely restored by IL-2 in the presence of IL-18. Thus, IL-18 enhances IFN-gamma production through an IL-12-dependent pathway and exhibits synergism when combined with IL-2 in terms of enhanced IL-13 and IFN-gamma production, suggesting the involvement of IL-18/IL-12/IL-2 pathway in modulating Th1/Th2 cytokine response.     

The T-786C NOS3 polymorphism in Alzheimer's disease: association and influence on gene expression

A common single nucleotide polymorphism (SNP), consisting in a T-->C transition (T-786C) in endothelial nitric oxide synthase (NOS3), has been reported to be associated with vascular pathologies, but no information are available on a possible association with AD. T-786C genotype was determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay in an Italian population of 432 AD patients compared with 360 healthy controls, matched for ethnic background, age and gender. Peripheral blood mononuclear cells (PBMC) from 22 subjects (11 AD and 11 controls) carrying different genotypes were isolated. Total RNA was extracted and analyzed by real-time PCR. No significant differences either in allelic or genotypic frequencies of the T-786C polymorphism between AD and normal population were observed, even stratifying AD patients by age at onset, gender, or ApoE status. However, expression of NOS3 in PBMC seems to be influenced by the presence of the C mutated allele, as demonstrated by a tendency towards a decrease in mRNA levels in C carriers, assessed by real-time PCR assay. This effect was observed both in patients and controls, independently from the cognitive impairment, and is likely to be dose-dependent, being mostly evident in CC homozygous. In conclusion, the T-786C SNP does not seem to be a risk factor for sporadic AD, but its presence correlates with a trend toward lower NOS3 expression rate, possibly exerting a beneficial effect in AD by contributing to lower oxidative damage.     

Imbalance between interleukin-6 and adrenomedullin mRNA levels in peripheral blood mononuclear cells of patients with lupus nephritis.

In this study, we measured the mRNA levels of adrenomedullin (AM), C-type natriuretic peptide, vascular endothelial growth factor, interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in peripheral blood mononuclear cells (PBMC) of 34 patients with lupus nephritis (LN) (15 active and 19 inactive) and 30 healthy volunteers. mRNA levels were measured using a real-time quantitative PCR METHOD: Compared with healthy volunteers, IL-6 mRNA levels were elevated in LN patients (P < 0.005), while AM mRNA levels were decreased (P < 0.05). Also, IL-6 mRNA levels were higher and AM mRNA levels lower in active LN patients compared with inactive LN patients. In addition, IL-6 mRNA levels positively correlated and AM mRNA levels negatively correlated with SLE disease activity index and laboratory findings, such as blood urea nitrogen, serum creatinine, 50% haemolytic unit of complement and urinary excretion of protein over 24 h. Furthermore, IL-6 mRNA levels were negatively correlated with AM mRNA levels within the same LN patients. With regard to pathological findings, our results showed that IL-6 mRNA levels were higher, and AM mRNA levels significantly lower in patients with a high activity index compared to those with a low activity index. Following treatment with prednisolone, IL-6 mRNA levels in active LN patients decreased and AM mRNA levels increased to levels comparable to those in inactive LN and healthy volunteers. In vitro studies further demonstrated that elevated IL-6 mRNA levels in active LN patient PBMC were suppressed by the addition of adrenomedullin. Our results suggest that an imbalance between IL-6 and AM levels may play an important role in the progression of SLE, and that the mRNA levels of these genes in PBMC may be used as a disease activity index for SLE.     

Effect of low-dose cyclosporin a microemulsion on disease severity, interleukin-6, interleukin-8 and tumor necrosis factor alpha production in severe pediatric atopic dermatitis

BACKGROUND: The release of cytokines [interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-alpha)] by skin cells is involved in the pathogenesis of atopic dermatitis (AD). Objective: To evaluate the effect of low-dose cyclosporin A (CyA) on clinical symptoms and cytokine secretion in severe pediatric AD. METHODS: Ten children with severe AD (SCORAD index >50) were treated for 8 weeks with CyA. The initial dose of 2.5 mg/kg/day was titrated to a maximum of 5 mg/kg/day until a SCORAD reduction of >or =35% was achieved ("treatment response"). After stopping CyA all patients entered a 4-week follow-up period. Cytokine secretion (IL-6, IL-8 and TNF-alpha) from patients' PBMC was assessed by ELISA before and after CyA treatment and was compared with 18 healthy nonatopic controls. Only the data of patients, who responded to CyA and did not experience a relapse during the follow-up period, were evaluated for this paper. RESULTS: Seven patients responded to CyA without relapse during the follow-up period. The median SCORAD index in these patients improved from 71 at baseline to 22 after CyA treatment (p < 0.001). AD patients' PBMC produced more IL-6, IL-8 and TNF-alpha than PBMC of controls. Suppression of IL-6 (p < 0.05) and IL-8 (p < 0.05) production was observed after CyA treatment. TNF-alpha levels were unchanged by CyA in all patients. CONCLUSIONS: The reduction in severity of pediatric AD with CyA is associated with decreased production of IL-6 and IL-8, but not TNF-alpha by PBMC.     

Effects of substance P and vasoactive intestinal peptide on interferon-gamma and interleukin-4 production in severe atopic dermatitis.

BACKGROUND: Recent studies have demonstrated that two T cell-derived lymphokines, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), play a crucial role in the pathogenesis of atopic dermatitis (AD). It is known that neuropeptides, such as substance P (SP) and vasoactive intestinal peptide (VIP), have various immunomodulatory effects. Elevated levels of these neuropeptides and increased staining of SP positive nerve fibers have been reported in AD patients. OBJECTIVE: The study was designed to examine the effects of SP and VIP on the production of IFN-gamma and IL-4. The aim of the study was to establish whether these neuropeptides acted to affect cytokine release in the peripheral blood mononuclear cells (PBMCs) of AD patients. METHODS: The effects of SP and VIP on the production of IFN-gamma and IL-4 in phytohemagglutinin stimulated PBMC cultures over a 48-hour period were analyzed by enzyme-linked immunosorbent assay in 15 AD patients. Non-atopic individuals were used as a control group. RESULTS: Base cytokine profiles of AD patients showed significantly decreased IFN-gamma and increased IL-4 when weighed against non-atopic controls. Compared with controls, SP had a significant percentage enhancing effect on both IFN-gamma and IL-4 production at concentrations of 10(-8) M and 10(-6) M, however, this IFN-gamma up-regulatory effect of SP was reversed by spantide, a SP antagonist. The ratios of IFN-gamma: IL-4 production were significantly elevated in the SP treated AD group. Although VIP had no specific noticeable effects on the IFN-gamma and IL-4 production. CONCLUSIONS: Our data may suggest that SP has an influence on the immunomodulation of AD patient by regulating IFN-gamma production, either directly or indirectly. Vasoactive intestinal peptide, on the other hand, has no modulatory effects on the cytokine production of AD patients.     

The skin fungus-induced Th1- and Th2-related cytokine, chemokine and prostaglandin E2 production in peripheral blood mononuclear cells from patients with atopic dermatitis and psoriasis vulgaris

BACKGROUND: It is suggested that skin fungi may be involved in the development of atopic dermatitis (AD) and psoriasis vulgaris (PV). OBJECTIVE: We studied skin fungus-induced Th1- or Th2-related cytokine, chemokine and prostaglandin E2 (PGE2) secretion in peripheral blood mononuclear cells (PBMC) from patients with AD and PV and normal subjects. METHODS: PBMC were cultured with the extracts of Malassezia furfur (MF), Candida albicans (CA) and Trichophyton rubrum (TR). The cytokine, chemokine and PGE2 amounts in the supernatants were measured by enzyme-linked immunosorbent assays. RESULTS: MF induced IL-4 and macrophage-derived chemokine (MDC) secretion in AD patients, while induced IFN-gamma and interferon-inducible protein of 10 kDa (IP-10) secretion in PV patients, however, did not induce either secretion in normal subjects. CA induced IL-4, MDC, IFN-gamma and IP-10 secretion in AD and PV patients and normal subjects. In AD patients, the magnitude of IL-4 and MDC responses to CA was higher than that to MF. Compared with PV patients and normal subjects, the magnitude of IL-4 and MDC responses to CA was higher while that of IFN-gamma and IP-10 responses to CA was lower in AD patients. TR induced moderate IL-4 and MDC secretion only in AD patients. The three fungi induced higher levels of PGE2 secretion in AD patients than in PV patients and normal subjects. Cyclooxygenase-2 inhibitor NS-398 suppressed PGE2 responses to MF, CA and TR, and partially suppressed IL-4 and MDC responses to MF, CA and TR, while enhanced IFN-gamma and IP-10 responses to CA in AD patients, and these effects of NS-398 were reversed by cyclic AMP analogue. CONCLUSION: AD patients manifest Th2-skewed responses to MF, CA and TR, which may be partially attributable to the enhanced PGE2 responses to these fungi. PV patients manifest Th1-skewed responses to MF.     

Correlation of Th1-type cytokine expression and induced proliferation to lipopolysaccharide

Recent evidence from mouse models indicates that neonatal exposure to lipopolysaccharide (LPS) can prevent experimentally induced allergic disease. Furthermore, we noted that human cord blood mononuclear cells (CBMC) have an increased proliferative response to LPS relative to their respective maternal peripheral blood mononuclear cells (PBMC). We sought, therefore, to examine the cytokine expression profile induced by LPS in CBMC and its relationship to the LPS-mediated proliferative response. CBMC and maternal PBMC were evaluated for IL-10, IL-4, IL-13, IL-12 alpha, and IFN-gamma expression after LPS stimulation by real-time PCR. IFN-gamma secretion was detected by enzyme-linked immunosorbent assay. LPS increased IFN-gamma and IL-13, but decreased IL-4 expression in CBMC (P < 0.024, P < 0.014, and P < 0.027, respectively). In PBMC, however, no significant changes in expression were noted after LPS stimulation. Stimulation by LPS significantly increased the secretion of IFN-gamma in CBMC compared with PBMC at the two concentrations analyzed (1 ng/ml, P < 0.048; 10,000 ng/ml, P < 0.003). The magnitude of the LPS-mediated proliferative effect in CBMC directly correlated to the level of induction of IFN-gamma (P < 0.01), but inversely correlated to the induced levels of IL-4 and IL-13 (P < 0.01 and P = 0.01, respectively). No association of the CBMC proliferative response to IL-12 alpha or IL-10 was noted. Thus, a high proliferative response to LPS in CBMC correlates with a change from a Th2- to Th1-induced cytokine expression profile. Since early exposure to LPS may protect against allergic disease, one may speculate that an aberrant response to LPS may increase the likelihood of developing overt disease in susceptible individuals.     

肺癌患者调节性T细胞的变化及CpG ODN的干预作用

目的:观察肺癌患者外周血调节性T细胞的变化,并探讨CpG ODN的干预作用。方法:分离肺癌患者和健康志愿者(各30例)的外周血单个核细胞(PBMC),用流式细胞仪检测CD4+CD25+调节性T细胞比例,Real-time PCR检测Foxp3基因的表达,ELISA法检测TGF-β和IFN-γ水平。将30例肺癌患者的PBMC随机分为实验组和安慰剂组,分别给予CpG ODN2006或安慰剂CpG ODN1612干预,比较干预前后上述指标的变化。结果:肺癌患者PBMC中的CD4+CD25+调节性T细胞比例、Foxp3基因的相对表达量、TGF-β水平均高于健康对照组,差异具有显著性,但不同病理分型和分期的肺癌患者亚组间比较无显著性差异。两组的IFN-γ水平比较无差异性。CpG ODN干预后CpG ODN2006治疗组的CD4+CD25+调节性T细胞比例、Foxp3基因的相对表达量及TGF-β水平出现了下降,差异有显著性,IFN-γ水平变化无显著性差异。而CpG ODN1612安慰剂组的上述指标在干预前后则无显著性改变。结论:肺癌患者外周血中的CD4+CD25+Foxp3+调节性T细胞比例、TGF-β水平明显升高。CpG ODN2006干预可下调CD4+CD25+Foxp3+调节性T细胞比例及TGF-β水平。