Interleukin-10 secretion from CD14+ peripheral blood mononuclear cells is downregulated in patients with acne vulgaris.

BACKGROUND: Acne is a common chronic inflammatory dermatosis of the pilosebaceous unit. It is characterized by seborrhoea, comedone formation and an inflammatory response consistent with defective cellular immunity to Propionibacterium acnes. OBJECTIVES: The objective of this study was to investigate the immune reactivity of patients with acne compared with healthy controls by examining the response of peripheral blood mononuclear cells (PBMCs) to stimulation with P. acnes. Particular focus was placed upon measuring the production of interleukin (IL)-10, which has an established immunoregulatory role. PATIENTS AND METHODS: Venous blood was collected from 47 patients with acne and 40 age- and sex-matched healthy controls with no prior history of acne. PBMCs were cultured and their cytokine response to P. acnes investigated. RESULTS: Proinflammatory IL-8 and tumour necrosis factor (TNF)-alpha secretion from PBMCs was higher in patients with acne when stimulated with P. acnes. In contrast, a statistically significant reduction in PBMC secretion of anti-inflammatory IL-10 in patients with acne was identified. The impaired production of IL-10 by PBMCs from patients with acne was confined to CD14+ cells presumed to be monocytes. The ability of CD14 cells from patients with acne to phagocytose P. acnes bacteria was also observed to be defective but the addition of exogenous IL-10 to PBMC cultures restored phagocytic activity. CONCLUSIONS: These data suggest that patients with acne have a proinflammatory cytokine milieu and crucially are unable to contain early inflammatory changes due to a specific defect in immunosurveillance, namely low monocyte IL-10 production. Our observations raise the possibility that acne therapeutics might profitably target IL-10 both as a regulator of proinflammatory cytokines and in augmenting the CD14+ cell phagocytic response.

Characterisation of the range of neutrophil stimulating mediators in cystic fibrosis sputum.

BACKGROUND: Most patients with cystic fibrosis (CF) die of respiratory failure due to chronic infection and destructive neutrophilic inflammation. OBJECTIVE: To identify potential therapeutic targets by characterising the neutrophil stimulating mediators in the CF airway. METHODS: Spontaneously expectorated CF sputum was extracted in phosphate buffered saline for assays of neutrophil chemotaxis, intracellular calcium mobilisation and cell shape change. Mediators were purified by ion exchange, C(18) reversed phase and size exclusion chromatography. RESULTS: A pool of CF sputum contained considerable neutrophil stimulating activity but neutralisation of interleukin (IL)8/CXCL8 had little inhibitory effect on neutrophil chemotactic (10149 (2023) migrating cells vs 8661 (2597) at 62 mg sputum/ml; NS) or shape change (% forward scatter increase 46 (8) vs 38 (5) at 19 mg sputum/ml; p<0.05) responses. Furthermore, the CF sputum pool induced an elevation in intracellular calcium ions even after desensitisation of the neutrophils to IL8. Chromatography identified contributions to the neutrophil shape change inducing activity from IL8, other CXC chemokines, leukotriene (LT) B(4) and two formyl peptides. There was also suggestive evidence for contributions from platelet activating factor (PAF) and C5a. Using non-chromatographed individual sputum samples, anti-IL8 alone did have an inhibitory effect on neutrophil chemotaxis (median inhibition 41%; p = 0.0002). However, even in this experiment, there were clearly significantly important, non-IL8 mediated, effects of CF sputum on neutrophils, and an inhibitor cocktail of anti-IL8 plus CXCR2, LTB(4), formyl peptide, PAF and C5a receptor antagonists inhibited chemotaxis by a median of 97% (p = 0.0002). CONCLUSION: Many chemoattractants contribute to the neutrophil stimulating activity in CF sputum although the relative contribution of these mediators differs in different patients. Selective blockade of single mediators may not be sufficient to control neutrophil recruitment and activation in the CF airway.

Retinoic acid inhibition of human papillomavirus type 16-mediated transformation of human keratinocytes.

We previously reported that human keratinocytes (HKc) immortalized by transfection with human papillomavirus type 16 DNA (HKc/HPV16) are more sensitive than normal HKc to growth inhibition by retinoic acid (RA), and that RA treatment of HKc/HPV16 inhibits HPV16 E6/E7 mRNA expression (L. Pirisi et al., Cancer Res., 52: 187-193, 1992). We now demonstrate that HPV16 E2 and E5 mRNAs are also decreased by RA treatment of HKc/HPV16, indicating a general inhibition by RA on the expression of HPV16 early genes. In addition, protein levels of E6 and E7, as measured by immunofluorescence, are also decreased in a dose-dependent manner following RA treatment of HKc/HPV16. Since E6 and E7 are considered the oncogenes of HPV16, we explored the possibility that RA may interfere with HPV16-mediated immortalization of HKc. RA treatment (1 nM) of normal HKc, immediately following transfection with HPV16 DNA, inhibited immortalization by about 95%. Overall, these results provide a direct biochemical basis for a role of RA in the chemo-prevention of human papillomavirus-induced cancers.

Increased sensitivity of human keratinocytes immortalized by human papillomavirus type 16 DNA to growth control by retinoids.

Human papillomavirus (HPV) type 16 (HPV16) is associated with a large percentage of cervical malignancies, and HPV16 DNA can immortalize human keratinocytes in vitro. The transforming ability of the virus resides primarily in the open reading frames E6 and E7. Retinoids are potent modulators of growth and differentiation of keratinocytes and have been shown to reverse cervical lesions resulting from HPV infection. We compared the sensitivity of normal human foreskin keratinocytes (HKc) and four immortalized HKc lines, independently obtained by transfection of different normal HKc strains with HPV16 DNA (HKc/HPV16), to growth control by retinoic acid (RA). All the HKc/HPV16 lines were 10- to 100-fold more sensitive than normal HKc to growth inhibition by RA in both clonal and mass culture growth assays. The precursor to RA, retinol, was also found to be a more potent inhibitor of growth of HKc/HPV16 than normal HKc, while beta-carotene did not inhibit growth of either normal HKc or HKc/HPV16. In addition, HKc/HPV16 lines were more sensitive than normal HKc to modulation of keratin expression by RA and retinol. No differences were observed in the rate of uptake of [3H]RA or [3H]retinol between normal HKc and HKc/HPV16. Dot blot analysis of RNA extracted from HKc/HPV16 cultured in the absence or in the presence of 10(-7) M RA showed that the expression of the HPV16 open reading frames E6 and E7 is reduced 2- to 4-fold by RA. In addition, Northern blot analysis demonstrated that RA inhibition of E6 and E7 expression was both dose and time dependent. Overall, these results suggest that the increased sensitivity of the HKc/HPV16 lines to growth control by RA may be mediated by an inhibition of the expression of HPV16 gene products which are required for the maintenance of continuous growth.

Human papillomaviruses and cervical neoplasia in South Carolina.

Human papillomaviruses (HPVs), particularly types 16, 18, and 33, have recently been suggested as etiological agents for cervical neoplasia. However, few studies have explored this relationship among low-income minority women. This case-control study of cervical intraepithelial neoplasia (CIN), detected by Pap smear screening among South Carolina women, investigates the association between HPV positivity and the cytological continuum of CIN. Cervical spatulas and cytobrushes used to collect Pap smears from all women attending health department family planning clinics in three coastal South Carolina counties were saved for subsequent HPV detection and typing. Among this cohort of approximately 6000 cervical samples collected from March through December 1991, those with CIN, atypia, and other cervical abnormalities and women with normal cervical cytology were identified. Women with CIN II or III (n = 28) were 21.9 times more likely to be HPV 16, 18, or 33 positive, while women with CIN I (n = 114) were 11.7 times more likely to be HPV 16/18/33 positive when compared with women having normal cervical cytology (n = 223) and adjusting for potential confounders. Women with atypia (n = 115) were 3.0 times more likely to be HPV 16/18/33 positive. A chi 2 test for trend in increasing HPV 16/18/33 prevalence with increasing severity of cervical lesions was highly significant (P = 0.0001). HPV 6 and 11 were not associated with CIN, nor was there a significant trend of increasing prevalence with increasing severity of cervical lesions. Worthy of further research is our finding that the overall prevalence of HPV positivity was low in this relatively high-risk population of low-income, primarily black women.

Fumonisin B1 induces apoptosis in cultured human keratinocytes through sphinganine accumulation and ceramide depletion.

Fumonisin B1 stimulates apoptosis in a variety of cell types and tissues. We examined the role of sphingolipid changes in fumonisin B1-stimulated apoptosis. Sphinganine accumulated rapidly, sphingosine levels remained unchanged, and ceramides decreased during fumonisin B1 exposure. Increased DNA fragmentation, decreased viability, and apoptotic morphology were observed in cells exposed to fumonisin B1, sphinganine, or N-acetylsphingosine. Co-exposure to N-acetylsphingosine or beta-chloroalanine, which blocks sphinganine accumulation, partially protected cells from fumonisin B1-induced apoptosis. These results illustrate three sphingolipid-dependent mechanisms for inducing apoptosis: accumulation of excess ceramide, accumulation of excess sphinganine, and depletion of ceramide or complex sphingolipids derived from ceramide.

Retinoic acid suppresses human papillomavirus type 16 (HPV16)-mediated transformation of human keratinocytes and inhibits the expression of the HPV16 oncogenes.

We have used a model system of normal HKc and HKc immortalized by transfection with HPV16 DNA (HKc/HPV16) to investigate the effect of RA on the growth of HKc/HPV16 and the expression of the HPV16 oncogenes E6 and E7. These studies found that HKc/HPV16 are about 100-fold more sensitive than normal HKc to growth inhibition by RA in both clonal and mass culture growth assays. The precursor to RA, retinol, was also found to be a more potent inhibitor of growth of HKc/HPV16 than normal HKc while beta-carotene did not inhibit growth of either normal HKc or HKc/HPV16. No differences were observed in the rate of uptake of [3H]RA or [3H]retinol between normal HKc and HKc/HPV16. Northern blot analysis of mRNA extracted from HKc/HPV16 cultured in the absence or in the presence of 10(-7) M RA showed that the expression of the HPV16 oncogenes E6 and E7 as well as the early ORFs E2 and E5 is substantially reduced following RA treatment. In addition, protein levels of E6 and E7, as measured by immunofluorescence (E6 and E7) and Western blot (E7) are also decreased by RA treatment of HKc/HPV16. Since E6 and E7 are considered the oncogenes of HPV16, we explored the possibility that RA may interfere with HPV16-mediated immortalization of HKc. The RA treatment (1 nM) of normal HKc, during or immediately following transfection with HPV16 DNA, inhibited immortalization by about 95%. Overall, these results provide a direct biochemical basis for a role of dietary retinoids in the chemoprevention of HPV-induced cancers.

Modulation of the host's immune response by schistosome larvae.

Schistosomes appear to have evolved several strategies to down-regulate the host's immune response in order to promote their own survival. For the host, down-regulation is also beneficial as it can limit the extent of pathology. It is widely accepted that schistosomes modulate the immune response during the chronic phase of infection after egg deposition has started. However, there is increasing evidence that modulation of the immune response can occur much earlier at the time infective cercariae penetrate the host skin. In this review, we explore the various lines of evidence that excretory/secretory (ES) molecules from cercariae down-regulate the host's immune response. We highlight the immunological factors that are produced and may be involved in regulating the immune system (e.g. IL-10, and eicosanoids), as well as speculating on possible mechanisms of immune modulation (e.g. mast-cell activation, T-cell apoptosis, and/or the skewed activation of antigen-presenting cells [APCs]). Finally, we draw attention to several molecules of schistosome origin that have the potential to stimulate the regulatory response (e.g. glycans) and link these to potential host receptors (e.g. TLRs and C-type lectins).

Regulation of plasminogen gene expression by interleukin-6.

Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and participates in other biological processes dependent upon proteolytic activity. Consequently, plasmin activity is tightly regulated by plasminogen activators and protease inhibitors. In this study, we examined whether regulation of plasminogen gene expression also might provide a new mechanism for controlling this system. We examined the effects of recombinant human interleukin-6 (rhIL-6), a pleiotropic cytokine, on plasminogen mRNA expression in primary murine hepatocytes and Hep3B human hepatoma cells. In primary hepatocytes, rhIL-6 and hydrocortisone separately increased plasminogen mRNA expression, but hydrocortisone did not markedly enhance the response to rhIL-6. Hep3B hepatoma cells exhibited more modest responses to rhIL-6. We used the polymerase chain reaction to amplify a 1,067-bp fragment of the human plasminogen promoter/5' flanking region. This fragment was cloned upstream of a luciferase reporter gene. Hep3B cells transiently transfected with this construct provided approximately 100-fold higher luciferase activity compared to cells transfected with control plasmids, and luciferase activity was increased approximately 4.5-fold when these cells were treated with rhIL-6. Furthermore, mice injected with rhIL-6 exhibited increases in hepatic plasminogen mRNA. Circulating plasminogen levels were significantly higher in the mice injected with rhIL-6 compared to mice injected with saline. Mice injected with lipopolysaccharide (an inducer of IL-6 in vivo) also showed increased hepatic plasminogen mRNA. Thus, plasminogen gene expression can be modulated by rhIL-6, suggesting a new mechanism for regulating biological systems that use plasmin.

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies.

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.

Sputum T lymphocytes in asthma, COPD and healthy subjects have the phenotype of activated intraepithelial T cells (CD69+ CD103+).

BACKGROUND: T cells of intraepithelial phenotype have previously been detected in bronchoalveolar lavage (BAL) fluid in a range of lung diseases; these cells express the adhesion molecule alpha(E)beta(7) integrin, CD103, the ligand for epithelial cell E-cadherin. In subjects with asthma CD4+ lymphocytes are the predominant T cell subtype found in bronchial biopsy specimens and in BAL fluid, whereas CD8+ lymphocytes have been shown to predominate in subjects with chronic obstructive pulmonary disease (COPD). The aim of this study was to analyse the expression of CD103, activation markers (CD25 and CD69), and chemokine receptors (CXCR3, CCR5 and CCR3) on CD4+ and CD8+ lymphocytes from sputum and peripheral blood of subjects with asthma, COPD, and healthy controls. METHODS: T cell surface markers were assessed by immunofluorescence labelling and flow cytometry of gated lymphocytes among CD45+ leucocytes in sputum cell suspensions. RESULTS: Sputum lymphocytes expressed higher levels of CD103 and CD69 than blood lymphocytes in all subject groups, with CD103 expressed at higher levels on CD8+ than on CD4+ cells. There were no detectable differences in numbers of CD4+ and CD8+ T cells between subjects with asthma, COPD and controls. The percentage of sputum lymphocytes expressing CXCR3 was lower in subjects with asthma or COPD than in healthy controls; CCR3 was not detectable on sputum or blood lymphocytes. CONCLUSIONS: Sputum T lymphocytes are predominantly of activated intraepithelial phenotype (CD103+ CD69+), and normal numbers of CD4+ and CD8+ T cell populations are found in the sputum of patients with asthma and COPD.