Effect of palmitic acid on neutrophil functions in vitro

BACKGROUND: It has been shown that in acne comedones the proportion of linoleic acid is markedly decreased, while palmitic acid is significantly increased. We previously reported that the decreased proportion of linoleic acid, which markedly suppresses neutrophil reactive oxygen species (ROS) generation and phagocytosis, contribute to the worsening of acne inflammation. The aim of this study was to examine the effect of palmitic acid on neutrophil functions in vitro. METHODS: We investigated the effect of palmitic acid on inflammatory parameters such as neutrophil chemotaxis, phagocytosis, and ROS generation. Reactive oxygen species generation in a cell-free, xanthine-xanthine oxidase system was also assessed. The species examined were superoxide radical anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH.). RESULTS: Palmitic acid significantly decreased H2O2 generation both by neutrophils and in the xanthine-xanthine oxidase system, while neutrophil chemotaxis and phagocytosis as well as O2- and OH. generation by both systems were not markedly affected in the presence of palmitic acid. CONCLUSIONS: The present study suggests that palmitic acid may be involved in the pathogenesis of acne inflammation from a standpoint of oxidative tissue injury.     

Effects of cepharanthin on neutrophil chemotaxis, phagocytosis, and reactive oxygen species generation.

The effects of cepharanthin on inflammatory parameters such as neutrophil chemotaxis, phagocytosis and reactive oxygen species (ROS) generation, were examined. Cepharanthin significantly decreased the levels of O2-, H2O2, and OH. generated by neutrophils. H2O2 and OH. generated in a cell-free, xanthine-xanthine oxidase system were also reduced in the presence of cepharanthin. However, the drug did not affect neutrophil chemotaxis or phagocytosis. The present study indicates that cepharanthin is an effective ROS scavenger, exerting its anti-inflammatory action by reducing the potent ROS species excessively generated in tissues and organs, especially at the sites of inflammation.     

Inhibitory effect of azelaic acid on neutrophil functions: a possible cause for its efficacy in treating pathogenetically unrelated diseases

Summary It has been shown that acne, hyperpigmentation and lentigo malignant are more or less related pathogenetically to reactive oxygen species (ROS). It has recently been reported that azelaic acid is effective in treating these conditions and that it possesses anti-enzymatic and anti-mitochondrial activity, including cytochrome-P₄₅₀ reductase and 5-reductase in microsomal preparations with nicotinamide adenine dinucleotide phosphate (NADPH). We therefore investigated the effects of azelaic acid on human neutrophil functions, such as chemotaxis, phagocytosis and ROS generation. ROS generation in a cell-free system was also assessed. The results revealed that neutrophil chemotaxis and phagocytosis as well as ROS generated in a xanthine — xanthine-oxidase system were not significantly changed in the presence of azelaic acid. However, azelaic acid markedly decreased O ₂ ^– and OH generated by neutrophils. It may be concluded that the reported clinical effectiveness of azelaic acid is partly due to its inhibitory action on neutrophil-generated ROS, leading to a reduction both in oxidative tissue injury at sites of inflammation and in melanin formation.     

Effects of subminimal inhibitory concentrations of minocycline on neutrophil chemotactic factor production in comedonal bacteria,neutrophil phagocytosis and oxygen metabolism

Summary Comedonal bacteria, Propionibacterium acnes, P. granulosum and coagulase-negative staphylococci (CNS) seem to play an important initiating role in the inflammatory process by producing neutrophil chemotactic factors. The attracted neutrophils, after phagocytosis, release inflammatory factors such as reactive oxygen species (ROS). We investigated the effects of minocycline at subminimal inhibitory concentrations (sub-MIC), i.e. one-tenth MIC, on the production of human neutrophil chemotactic factors in comedonal bacteria, and on several inflammatory parameters of neutrophils, including neutrophil phagocytosis and generation of ROS (O ₂ ^– , H₂O₂, O ). ROS generation in a cell-free, xanthinexanthine oxidase system was also assessed. Production of neutrophil chemotactic factors in all strains of P. acnes, P. granulosum and CNS were significantly suppressed by sub-MIC minocycline. Sub-MIC minocycline effectively reduced three kinds of neutrophil-generated ROS (O ₂ ^– , H₂O₂, O ). However, neutrophil phagocytosis and the ROS generated in a cell-free system were not markedly changed in the presence of sub-MIC minocycline. The results suggest that sub-MIC minocycline has an anti-inflammatory effect by inhibiting the production of neutrophil chemotactic factors in comedonal bacteria as well as ROS generated by neutrophils in the inflammatory process of acne.     

Increased hydrogen peroxide generation by neutrophils from patients with acne inflammation

BACKGROUND: Reactive oxygen species generated by neutrophils are closely correlated with the pathogenesis of a variety of inflammatory skin diseases. The aim of this study was to investigate the possible role of reactive oxygen species generated by neutrophils in the mediation of acne inflammation. METHODS: Bacterial phagocytotic stimuli, mediated by opsonin activity, were applied to whole blood, and neutrophil hydrogen peroxide production was measured. RESULTS: Patients with acne inflammation showed a significantly increased level of hydrogen peroxide produced by neutrophils compared to patients with acne comedones and healthy controls. There were no marked differences in the level of hydrogen peroxide produced by neutrophils between patients with acne comedones and healthy controls. In addition, patients with acne inflammation treated by oral administration of minocycline hydrochloride, a drug that inhibits hydrogen peroxide generation by neutrophils, showed a significant decrease in the ability of neutrophils to produce hydrogen peroxide in accordance with a decrease in the inflammatory activity of acne lesions. CONCLUSIONS: The present study seems to suggest that acne inflammation is mediated in part by hydrogen peroxide generation by neutrophils.     

The inhibition of free radical generation by human neutrophils through the synergistic effects of metronidazole with palmitoleic acid: a possible mechanism of action of metronidazole in rosacea and acne

Summary Metronidazole is clinically effective in treating not only rosacea but also acne inflammation. Yet it is generally considered not to be very effective in inhibiting the growth of anaerobic Propionibacterium acnes. We report here our investigation into the synergistic effects of metronidazole and palmitoleic acid on the anaerobic growth of P. acnes as well as on human neutrophil functions, including the generation of reactive oxygen species (ROS). Both metronidazole and palmitoleic acid, when used alone, only slightly inhibited the growth of P. acnes, and no significant decrease in human neutrophil functions, including the generation of ROS, was observed. But metronidazole used in the presence of palmitoleic acid markedly inhibited the anaerobic growth of P. acnes and decreased ROS generation by neutrophils. However, ROS generated in the xanthine-xanthine oxidase system were not affected. Metronidazole was shown to be clinically effective by decreasing neutrophil-generated ROS at the sites of inflammation with the aid of palmitoleic acid, which is generally present in human skin. By inhibiting oxidative tissue injury under in vivo conditions, treatment with metronidazole results in remarkable improvement of rosacea and acne.     

Impaired neutrophil functions in patients with leukocytoclastic vasculitis

Background Leukocytoclastic vasculitis (LV) is characterized by segmental inflammation of small blood vessels, resulting in ischemic damage to the surrounding tissue. It is considered to be related to a type III hypersensitivity reaction, although the exact etiologic mechanism is not clear. Objective The purpose of this study was to evaluate neutrophil functions in patients with LV in order to understand their role in the pathogenesis of the disease. Methods Neutrophil functions were examined in 25 LV patients. The patients were divided into two groups: Group A consisted of 14 patients with drug-induced LV and Group B consisted of 11 patients where LV was induced by other factors. Results Both groups of patients showed significantly reduced chemotaxis and phagocytosis. Superoxide generation was significantly lower (P < 0.001) only in neutrophils from patients in Group A: 5.8 ± 0.5 nmoles O₂/10⁶ cells/min compared to 9.08 ± 0.8 nmoles O2/106 cells/min in the controls. Preincubation on normal neutrophils with the patients' sera caused an increase in their superoxide generation in accordance with the high IL-8 levels in these sera. Conclusions Neutrophil functions were significantly impaired in patients with LV. It is likely that factors present in LV plasma may chronically activate neutrophils, so that they become refracfory to further stimulation. Our study showed that neutrophil superoxide generation is low only in drug-induced LV; this test may assist in distinguishing such patients from those with LV induced by other causes.     

Effect of doxycycline on the generation of reactive oxygen species: a possible mechanism of action of acne therapy with doxycycline.

On the basis of a recent report that minocycline is effective in the treatment of acne inflammation by acting directly as an antioxidant on infiltrating neutrophils, we investigated whether doxycycline might also be capable of reducing the generation of reactive oxygen species, using human neutrophils and a cell-free, xanthine-xanthine oxidase system. The species investigated are superoxide radical anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH.). Doxycycline significantly reduced the levels of O2-, H2O2 and OH. generated by both systems. Our results seem to suggest that the clinical effectiveness of doxycycline in the treatment of acne inflammation is due partly to its antioxidant effect on neutrophils.     

Factors secreted by untreated psoriatic monocytes enhance neutrophil functions

Monocytes stimulated with bacterial lipopolysaccharides (LPS) release mediators that induce increased responses of human granulocytes. Recently we showed that psoriatic monocytes can stimulate neutrophil chemotaxis, phagocytosis, and O2- production without addition of LPS and this effect is inhibited by cyclosporin A. We have now investigated the presence of cytokines in supernatants from cultures of psoriatic monocytes (resting monocytes). These cells were cultured for 24 h in endotoxin-free medium. Normal human neutrophils were then incubated for 1 h with the resulting supernatants (sMS, or conditioned media). The sMS from unstimulated psoriatic monocytes significantly enhanced neutrophil chemotaxis and superoxide anion production. The enhancing factors are protein in nature and require ongoing protein synthesis, demonstrated by the facts that the activity in conditioned medium is labile to heat denaturation at 100 degrees C for 10 min, is not produced by monocytes cultured in the presence of puromycin, and is proteinase sensitive. Additional evidence suggested that extremes of pH inhibit activity. None of the conditioned media treated in these ways activated neutrophils. The neutrophil function-enhancing factors derived from psoriatic monocytes are in part cytokines, including TNF and GM-CSF. The support for this conclusion is the higher level of TNF and GM-CSF in media conditioned by psoriatic monocytes than in media conditioned by normal human monocytes, the inhibition of TNF production and neutrophil stimulating activity by cyclosporin A, and the inhibition of neutrophil stimulating activity in conditioned media preincubated with anti-TNF and anti-GM-CSF antibodies. It is concluded that psoriatic monocytes spontaneously produce higher than normal levels of TNF alpha, GM-CSF, and, perhaps, other cytokines that might be responsible for the enhanced activity of psoriatic neutrophils.     

SUCCESSFUL TREATMENT OF A PATIENT WITH RECURRENT FURUNCULOSIS BY VITAMIN C: IMPROVEMENT OF CLINICAL COURSE AND OF IMPAIRED NEUTROPHIL FUNCTIONS

Background. Neutrophils play a critical role in host defense against a variety of microbial pathogens. There is much information to suggest a role for vitamin C in the physiology of neutrophils. Thus, the effects of vitamin C treatment were studied in a patient with a history of recurrent furunculosis who showed altered neutrophil functions. Methods. Superoxide generation was measured by cytochrome C reduction. Phagocytosis of opsonized zymosan by neutrophils and chemotaxis on agarose plates were determined. Results. Chemotaxis, phagocytosis, and superoxide generation of the patient's neutrophils were significantly lower than those of the matched control. Treatment with vitamin C (500 mg/day) for 30 days caused a dramatic clinical response and a significant improvement of all three neutrophil functions to values similar to those of the controls. Conclusions. We suggest that the patient described here had a temporary defect in neutrophil functions. The treatment with vitamin C probably prevented neutrophil oxidation, thus contributing to recovery of neutrophil function and arrest of furunculosis.     

Neutrophil functions in acne conglobata.

Chemotaxis, random migration, phagocytosis, spontaneous and stimulated NBT reduction were evaluated in neutrophils from the peripheral blood of 8 patients with acne conglobata and were found to be normal. Only 2 patients had an increased spontaneous NBT reduction, and another exhibited a defect in neutrophil adhesiveness. On this basis, intrinsic defects of the major neutrophil functions, as evaluated in vitro, can be excluded in acne conglobata.     

Increased plasma chemoattractant in Sweet''s syndrome

The neutrophil function and plasma leukotactic activity of a patient with Sweet's syndrome and cystonodular acne were evaluated during a 2 1/2-year period. These studies demonstrated that chemotaxis was frequently slightly increased, especially during an exacerbation of Sweet's syndrome, but showed some decrease during isotretinoin therapy. Other functions, such as phagocytosis, metabolic activation, and bacterial killing, also were slightly increased. In addition, the patient's serum contained a heat-stable, nonlipid chemoattractant that was present at all times except during a course of isotretinoin. Although his symptoms responded to aspirin, the plasma continued to show this chemoattractant. These findings are consistent with the hypothesis that excess chemoattractant in Sweet's syndrome attracts neutrophils, which then mediate an inflammatory response. In addition, aspirin may be used to control Sweet's syndrome symptoms, although it does not suppress the plasma chemoattractant.     

Isotretinoin produces significant inhibition of monocyte and neutrophil chemotaxis in vivo in patients with cystic acne

The effect of oral isotretinoin (13-cis-retinoic acid) on in vivo chemotactic responses was studied longitudinally in 7 patients with cystic acne. As measured in a microchamber chemotaxis assay, both monocyte and neutrophil chemotaxis were inhibited 98% (p less than 0.001) during isotretinoin treatment. In vivo chemotactic responses returned to normal within 2 months of cessation of treatment. Biopsies of skin chamber sites from patients on isotretinoin showed no significant dermal or epidermal leukocytic accumulation in response to autologous zymosan-activated serum, whereas chambers from controls showed extensive neutrophilic infiltrates even in the epidermis. In contrast, in vitro chemotactic responses of neutrophils and monocytes from patients on isotretinoin were not diminished. Sera and plasma from patients on isotretinoin contained no inhibitors of chemotaxis, and activated sera from these patients were excellent attractants for normal monocytes. We postulate that isotretinoin produces significant anti-inflammatory effects by inhibition of monocyte and neutrophil chemotaxis across intact biologic barriers in vivo.     

Increased advanced oxidation protein products in Behçet's disease: a new activity marker?

Background Behçet's disease (BD) is a chronic inflammatory disease with unknown pathogenesis. As various functions of neutrophils in peripheral blood, such as chemotaxis, phagocytosis and generation of reactive oxygen species (ROS) increase in BD, ROS‐mediated oxidative stress related to neutrophil activation may have an important role in the pathogenesis of BD. Objectives To investigate the importance of neutrophil activation as the main source of oxidative stress through protein oxidation in the pathogenesis of BD, and also to investigate whether one of the products of protein oxidation, advanced oxidation protein products (AOPP), may be used as an activity marker for BD. Methods Patients with BD (n = 49), at active and inactive stages, with or without evidence of uveitis, and healthy volunteers (n = 40) were entered into the study. A full blood count, peripheral blood smears, routine biochemical analyses, C‐reactive protein (CRP) and erythrocyte sedimentation rate (ESR) measurements were performed in all patients preceding the study. Plasma myeloperoxidase (MPO) activity, representing neutrophil activation, and biomarkers of oxidative stress reflecting protein oxidation, such as levels of AOPP and thiol, were measured spectrophotometrically. Statistical comparisons were made using Mann–Whitney U‐tests, Student's t‐tests, anova /post‐anova tests and correlation analyses. Results In all patients, the results of full blood count, peripheral blood smears and routine biochemical analyses were in the normal range, but mean values of CRP and ESR were higher than laboratory reference values. Plasma MPO activity and AOPP levels were found to be higher and thiol values lower in the total patient group and individual subgroups than in controls. Patients with active BD had significantly higher MPO and AOPP levels and lower thiol levels than patients with inactive BD. There was no difference between uveitis‐positive and uveitis‐negative subgroups in MPO and thiol levels, but AOPP levels were lower in the latter group. Patients with active BD ± uveitis were shown to have increased MPO and AOPP but decreased thiol levels in comparison with the inactive BD, uveitis‐negative subgroup. There were strong positive correlations between ESR and CRP, ESR and MPO, ESR and thiol, ESR and AOPP, CRP and MPO, CRP and AOPP, MPO and AOPP, and thiol and AOPP levels in patients with BD. Conclusions Based on this first study, in which MPO‐mediated AOPP formation has been demonstrated, it may be suggested that activated neutrophils may play an important role in the pathogenesis of BD and that chlorinated oxidants of neutrophil origin may lead to oxidative stress, notably protein oxidation. Therefore, AOPP may be a useful marker for monitoring the progress and the severity of the disease activity.     

Cigarette Smoke-Induced Interleukin-1 Alpha May Be Involved in the Pathogenesis of Adult Acne

Background

Lipid peroxide (LPO) in comedones, which are produced as a result of sebum oxidation, might potentially induce interleukin-1α (IL-1α) and exacerbate comedogenesis and inflammatory changes in comedones.

Objective

To investigate the relationship of proinflammatory cytokines and LPO levels in the extracts of comedones with the acne of clinical difference between smokers and non-smokers, and with the severity and distribution of the acne lesions.

Methods

Twenty-two non-smoking and 21 smoking adult acne patients were evaluated by comedone extraction and measurement of proinflammatory cytokines and LPO levels. Acne severity and distribution of the lesions were also analyzed.

Results

Relative to the non-smoking group, smokers had significantly higher levels of IL-1α and LPO in comedones. Their levels showed a positive correlation. However, there were no statistically significant difference between the severity or distribution of the disease and the levels of LPO and IL-1α in comedones.

Conclusion

Smoking may be involved in the pathogenesis of adult acne by increasing the oxidative stress that results in subsequent accumulation of LPO in comedones.     

The clinical features of late onset acne compared with early onset acne in women

Background Little is known about the clinical characteristics of acne based on the age of onset. Objectives The aim of this study was to investigate the clinical characteristics of patients according to the age of onset of acne and evaluate whether the findings were related to regional differences in the density of Propionibacterium acnes or the levels of sebum secretion. Methods A total of 89 women were recruited. The acne lesions were assessed by counting the lesions using standard digital photographs. Digital fluorescent photography for the evaluation of the density of P. acnes were taken and quantitative measurements of facial sebum secretion were performed. Results In women with acne, the age of onset was negatively correlated with the number of comedones and the proportion of comedones. By comparing the number of comedones and the proportion of comedones, onset of acne after 21 years of age was defined as late onset acne. In the patients with late onset acne, the number of comedones, the total number of acne lesions and the proportions of comedones were significantly less than in the patients with early onset acne. However, there were no significant differences in the fluorescence density of P. acnes or the level of sebum secretion between the two groups. Conclusions The results of this study, using objective evaluation tools, suggest that late onset acne has different clinical characteristics. Other possible factors might explain the clinical differences in late onset acne.     

Anti-inflammatory effects of erythromycin and tetracycline on Propionibacterium acnes induced production of chemotactic factors and reactive oxygen species by human neutrophils

Propionibacterium acnes (P. acnes), an anaerobic pathogen, plays an important role in the pathogenesis of acne and seems to initiate the inflammatory process by producing neutrophil chemotactic factors (NCF). Once neutrophils attracted by bacterial chemoattractants reach the inflamed site, they release inflammatory mediators such as lysosomal enzymes and reactive oxygen species (ROS). Previously, it has been shown that antibiotics may affect acne by means other than their anti-bacterial effects. Thus, we investigated the effect of subminimal inhibitory concentration (sub-MIC) of tetracycline and erythromycin on production of NCF and ROS. NCF was tested in vivo in a mouse model and ROS was estimated on human PMNL in vitro, by nitroblue tetrazolium dye reduction test (NBT) and cytochrome-C reduction test. Tetracycline (CS-T) and Erythromycin (CS-E) treated cultures showed a significant reduction of 35.8% and 58.3% in NCF production respectively, as compared to P. acnes stimulated cultures. Tetracycline and erythromycin at their sub-MIC also significantly inhibited release of ROS from human PMNL. Thus, tetracycline and erythromycin, besides having antibacterial activity, also have an anti-inflammatory action. These antibiotics reduce the capacity of P. acnes to produce NCF, as well decrease its ability to induce ROS from PMNL.     

申克孢子丝菌酵母相与白念珠菌诱导中性粒细胞活性氧簇的差异性表达

目的 研究人中性粒细胞吞噬申克孢子丝菌酵母相和白念珠菌后胞内活性氧簇（ROS）的实时表达水平及对两者杀菌能力的差异。 方法 应用密度梯度离心法分离人外周血中性粒细胞,并通过ROS探针（DCFH-DA）、流式细胞仪和激光共聚焦显微镜检测申克孢子丝菌酵母相临床株和白念珠菌标准株ATCC 90028作用后,中性粒细胞内ROS的实时表达水平及对上述2种真菌孢子的杀菌率。 结果 申克孢子丝菌酵母相临床株和白念珠菌标准株ATCC 90028作用中性粒细胞60 min后,申克孢子丝菌组中性粒细胞内ROS表达水平达到峰值（平均荧光强度159.67 ± 11.34）,并显著高于白念珠菌组（112.22 ± 9.66）,经LSD-t检验,P < 0.01;而作用120 ～ 180 min后,胞内ROS表达水平（120 min为89.01 ± 9.81;180 min为57.63 ± 8.46）迅速下降,显著低于白念珠菌组中ROS的同期表达水平（120 min时为110.25 ± 7.28;180 min时为109.98 ± 9.00,经LSD-t检验,120 min时P < 0.05,180 min时P < 0.01）。激光共聚焦显微镜观察到,ROS主要表达于吞噬上述真菌孢子的中性粒细胞中,并被募集到被吞噬的部分孢子表面。中性粒细胞对申克孢子丝菌180 min杀菌率（19.21% ± 3.68%）亦显著低于其对白念珠菌孢子的杀菌率（26.63% ± 4.97%）,经LSD-t检验,P < 0.01。 结论 中性粒细胞吞噬上述2种真菌孢子后,胞内ROS呈明显的差异性表达。与白念珠菌相比,中性粒细胞内ROS水平的迅速下降在一定程度上抑制了其对申克孢子丝菌的杀伤效能。 【关键词】 孢子丝菌属; 念珠菌,白色; 中性白细胞; 活性氧     

Neutrophil chemotaxis in psoriasis.

Neutrophil chemotaxis was assessed in 69 psoriatic patients and 37 healthy human subjects. It was found to be significantly enhanced in 52 untreated patients. In 20 patients treated with an orally-administered phosphodiesterase inhibitor, Diphylline, neutrophil chemotaxis was normal. The enhanced chemotactic response of neutrophils from untreated patients with minimal skin lesions was at least equal to the response of those from patients with extensive skin lesions. Preincubation of normal human leukocytes with plasma derived from patients with widespread lesions markedly reduced their chemotactic activity. Plasma derived from patients with extensive skin lesions exhibited marked chemoattracting properties in comparison with plasma from healthy subjects. It is postulated that the basic intrinsic abnormality of neutrophil function in psoriasis could be caused by a decreased cyclic AMP/cyclic GMP ratio, similar to the decreased cyclic AMP/cyclic GMP ratio found in the lesional epidermis of this disease. Plasma factors which influence chemotaxis in psoriasis are related to the extent of the eruption and their effect is contrary to the effect of the basic intrinsic abnormality of psoriatic neutrophils.     

Changes in laboratory variables induced by isotretinoin treatment of acne

During a trial of isotretinoin (0.5 mg/kg body weight/day for 3 months) in 90 patients with severe acne, the leucocyte (WBC) count, and particularly the number of neutrophils, decreased significantly. In patients with a good response the mean WBC count fell by 24% and the neutrophils by 33%, whereas in those with a poor response these variables decreased by 8% and 14%, respectively. The serum ALAT, ASAT, cholesterol and triglyceride levels increased significantly. Patients with a poor response (n = 35) received a higher dosage (0.75 mg/kg) for an additional 3 months, and during this period there was a further decrease in the WBC and neutrophil counts and an increase in the triglyceride level. In the other patients, who initially responded well, the dosage was decreased to 0.1 or 0 mg/kg during the second 3-month period, which resulted in reversion of the laboratory variables to the pre-treatment levels. The observed changes were clearly both dose-dependent and reversible.