Regulation of human neutrophil oxidative burst by pro- and anti-inflammatory cytokines

Human polymorphonuclear neutrophils play a key role in host defenses against invading microorganisms. In response to a variety of stimuli, neutrophils release large quantities of superoxide anion (O2.-) in a phenomenon known as the respiratory burst. O2.- is the precursor of potent oxidants, which are essential for bacterial killing and also potentiate inflammatory reactions. Regulation of this production is therefore critical to kill pathogens without inducing tissue injury. Neutrophil production of O2.- is dependent on the respiratory burst oxidase, or NADPH oxidase, a multicomponent enzyme system that catalyzes NADPH-dependent reduction of oxygen to O2.-. NADPH oxidase is activated and regulated by various neutrophil stimuli at infectious or inflammatory sites. Proinflammatory cytokines such as GM-CSF, TNF and IL-8 modulate NADPH oxidase activity through a priming phenomenon. These cytokines induce a very weak oxidative response by PMN but strongly enhance neutrophil release of reactive oxygen species on exposure to a secondary applied stimulus such as bacterial N-formyl peptides. Priming phenomena are involved in normal innate immune defense and in some inflammatory diseases. The mechanisms underlying the priming process are poorly understood, although some studies have suggested that priming with various agonists is regulated at the receptor and post-receptor levels. Resolution of inflammation involves desensitization phenomena and cytokines are involved in this process by various mechanisms. A better understanding of phenomena involved in the regulation of NADPH oxidase could help to develop novel therapeutic agents for inflammatory diseases involving abnormal neutrophil superoxide production.     

Differential effects of short-chain fatty acids on proliferation and production of pro- and anti-inflammatory cytokines by cultured lymphocytes

Short-chain fatty acids (SCFA) are produced by fermentation of water-soluble fiber by anaerobic bacteria in the large bowel. Fiber-rich diets decrease the risk of developing inflammatory bowel disease (IBD) and butyrate enemas are effective as a therapy in some patients. Crohn's disease, one form of IBD, appears to involve an exagerated T helper-1 (Th1) lymphocyte phenotype, characterised by production of interleukin (IL)-2 and interferon (IFN)-gamma, that drives the inflammation. To examine whether SCFA influence pro- and anti-inflammatory cytokine production, rat mesenteric lymph node lymphocytes were cultured in the presence of acetate (10 mM), butyrate (1.5 mM) or propionate (2 mM) and the production of cytokines in response to concanavalin A determined. Butyrate, but not acetate or propionate, inhibited lymphocyte proliferation and IL-2 production. Acetate and propionate were able to partly prevent the inhibitory effect of butyrate on IL-2 production. Acetate and propionate increased IFN-gamma production, whereas butyrate inhibited it. Acetate and propionate in combination were able to prevent the inhibitory effect of butyrate on IFN-gamma production. IL-4 was not detected in any cultures. Acetate and propionate increased IL-10 production, which was not affected by butyrate. It is concluded that butyrate significantly inhibits Th1-type responses and that this might explain the therapeutic effect of butyrate in IBD patients. Acetate and propionate have less marked modulatory actions, and in some cases have effects that oppose those of butyrate. A combination of the three SCFA causes a shift in the T helper lymphocyte phenotype towards a more anti-inflammatory phenotype and this might explain the protective effects of fiber.     

In vitro modulation of human keratinocyte pro- and anti-inflammatory cytokine production by the capsule of Malassezia species

Malassezia spp. are commensal, cutaneous fungi that are implicated in seborrhoeic dermatitis. We hypothesize that the lipid-rich capsule of Malassezia spp. masks the organism from host detection, and depletion of this layer elicits an inflammatory response. To test this, preparations of capsulated or acapsular [10% (v/v) Triton X-100 treated], viable and nonviable, exponential or stationary phase Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia restricta, Malassezia slooffiae and Malassezia sympodialis, were incubated with normal human keratinocytes. Proinflammatory (IL-6, IL-8, IL-1alpha and tumour necrosis factor-alpha) and anti-inflammatory cytokine (IL-10) release and intracellular IL-10 concentrations were quantified using enzyme-linked immunosorbent assays. Capsulated Malassezia yeasts stimulated limited or no production of inflammatory cytokines, and increased intracellular IL-10 (P < 0.05). Removal of the capsule of many Malassezia preparations caused a significantly increased production of IL-6, IL-8 and IL-1alpha, and a decrease in intracellular IL-10. Notably, acapsular viable, stationary phase M. globosa caused a 66-fold increase in IL-8 production (P < 0.001) and acapsular nonviable, stationary phase M. furfur caused a 38-fold increase in IL-6 production (P < 0.001) and a 12-fold decrease in intracellular IL-10 (P < 0.001). These results support the hypothesis that the lipid layer of Malassezia spp. modulates cytokine production by keratinocytes. This has implications in the pathogenesis of seborrhoeic dermatitis.     

Abnormal production of pro- and anti-inflammatory cytokines by lupus monocytes in response to apoptotic cells

Monocytes are a key component of the innate immune system involved in the regulation of the adaptive immune response. Previous studies have focused on apoptotic cell clearance abnormalities in systemic lupus erythematosus (SLE) monocytes. However, whether SLE monocytes might express unique patterns of cytokine secretion in response to apoptotic cells is still unknown. Here, we used monocytes from healthy controls and SLE patients to evaluate the production of TNF-α and TGF-β in response to apoptotic cells. Upon recognition of apoptotic material, monocytes from healthy controls showed prominent TGF-β secretion (mean ± SD: 824.6±144.3 pg/ml) and minimal TNF-α production (mean ± SD: 32.6±2.1 pg/ml). In contrast, monocytes from SLE patients had prominent TNF-α production (mean ± SD: 302.2±337.5 pg/ml) and diminished TGF-β secretion (mean ± SD: 685.9±615.9 pg/ml), a difference that was statistically significant compared to normal monocytes (p≤10⁻⁶ for TNF-α secretion, and p = 0.0031 for TGF-β, respectively). Interestingly, the unique cytokine response by SLE monocytes was independent of their phagocytic clearance efficiency, opsonizing autoantibodies and disease activity. We further showed that nucleic acids from apoptotic cells play important role in the induction of TNF-α by lupus monocytes. Together, these observations suggest that, in addition to potential clearance defects, monocytes from SLE patients have an abnormal balance in the secretion of anti- and pro-inflammatory cytokines in response to apoptotic cells. Since the abnormal cytokine response to apoptotic material in SLE is not related to disease activity and opsonizing autoantibodies, it is possible that this response might be an intrinsic property of lupus monocytes. The studies focus attention on toll-like receptors (TLRs) and their downstream pathways as mediators of this response.     

Enterovirus-induced production of pro-inflammatory and T-helper cytokines by human leukocytes

Enteroviruses are associated with chronic inflammatory and autoimmune diseases in humans. In these conditions, the cytokine network is supposed to have an important role in inflammation and modulation of the (auto)immune response. In the present study, we demonstrate that coxsackie virus B4 and poliovirus type 1 induce production of pro-inflammatory cytokines IL-1 beta and TNF-alpha in freshly isolated human leucocytes. Furthermore, enteroviruses stimulate the production of cytokines belonging to Th(1)pathways (IFN-gamma, IL-2), and IL-10, which play a role in regulation of the cellular and humoral immune response.     

Differential effects of pro- and anti-inflammatory cytokines alone or in combinations on the metabolic profile of astrocytes

We have previously reported that the pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) induce profound modifications of the metabolic profile of astrocytes. The present study was undertaken to further characterize the effects of cytokines in astrocytes and to determine whether similar effects could also be observed in neurons. To do so, selected pro-inflammatory (IL-6 and interferon-γ, in addition to the above-mentioned TNFα and IL-1β) and anti-inflammatory cytokines (IL-4, IL-10, transforming growth factor-β1 and interferon-β) were applied to primary neuronal and astrocytic cultures, and key metabolic parameters were assessed. As a general pattern, we observed that pro-inflammatory cytokines increased glucose utilization in astrocytes while the anti-inflammatory cytokines IL-4 and IL-10 decreased astrocytic glucose utilization. In contrast, no significant change could be observed in neurons. When pairs of pro-inflammatory cytokines were co-applied in astrocytes, several additive or synergistic modifications could be observed. In contrast, IL-10 partially attenuated the effects of pro-inflammatory cytokines. Finally, the modifications of the astrocytic metabolism induced by TNFα?+?IL-1β and interferon-γ modulated neuronal susceptibility to an excitotoxic insult in neuron-astrocyte co-cultures. Together, these results suggest that pro- and anti-inflammatory cytokines differentially affect the metabolic profile of astrocytes, and that these changes have functional consequences for surrounding neurons.     

Production of pro- and anti-inflammatory cytokines by monocytes from patients with paracoccidioidomycosis

Monocytes and macrophages can produce a large repertoire of cytokines and participate in the pathogenesis of granulomatous diseases. We investigated the production of pro- and anti-inflammatory cytokines by monocytes from patients with active paracoccidioidomycosis. Peripheral blood monocytes from 37 patients and 29 healthy controls were cultivated with or without 10 microg/ml of lipopolysaccharide (LPS) for 18 h at 37 degrees C, and the cytokine levels were determined in the culture supernatants by enzyme immunoassay. The results showed that the endogenous levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-8, IL-10 and transforming growth factor beta detected in the supernatant of patient monocytes cultivated without stimulus were significantly higher than those produced by healthy controls. These data demonstrated that monocytes from patients with active paracoccidioidomycosis produce high levels of cytokines with both inflammatory and anti-inflammatory activities. However, patient monocytes produced significantly lower TNF-alpha and IL-6 levels in response to LPS when compared to normal subjects, suggesting an impairment in their capacity to produce these cytokines after LPS stimulation. Concentrations of IL-1beta, IL-8 and IL-10 in cultures stimulated with LPS were higher in patients than in controls. These results suggest that an imbalance in the production of pro- and anti-inflammatory cytokines might be associated with the pathogenesis of paracoccidioidomycosis.     

Adiponectin induces the anti-inflammatory cytokines IL-10 and IL-1RA in human leukocytes

There is increasing evidence that the adipose tissue and immunologic processes are closely linked. The most abundant protein within the adipocyte is adiponectin. Our current work reports that adiponectin has potent immuno-suppressive properties, as it induces the production of the anti-inflammatory mediators IL-10 and IL-1RA in primary human monocytes, monocyte-derived macrophages, and dendritic cells. In addition, adiponectin significantly impaired the production of the pro-inflammatory cytokine IFN-gamma in human macrophages. Moreover, adiponectin-treated macrophages exhibit a reduced phagocytotic and allo-stimulatory capacity. However, we could not detect any functional deficits or phenotypic changes in adiponectin-treated monocytes and monocyte-derived DC. In summary, the presented data support the idea that adiponectin might be of critical relevance for cytokine regulation in obesity and fatty liver diseases affecting primarily macrophage functions. This might represent a fundamental link between over-nutrition and an impaired inflammatory immune response.     

Relation of pro- and anti-inflammatory cytokines and the production of nitric oxide in patients receiving high-dose immunotherapy with interleukin-2

Immunotherapy with intravenous recombinant human interleukin-2 (rh IL-2) may be accompanied by hypotension and the emergence of capillary leak syndrome. Nitric oxide (NO) is supposed to be responsible for both side effects. The aim of the current investigation was to elucidate the relationship between pro- and anti-inflammatory cytokines and the production of NO in eight tumor patients receiving intravenous rh IL-2 continuously over a time period of 120 hours. Markers of systemic inflammation, as well as nitrate plasma levels, were consecutively determined. Significant changes in the levels of pro-inflammatory cytokines IL-6 and IL-8 were observed (p < 0.05). In contrast to the anti-inflammatory cytokine IL-10, which did not increase significantly, the serum concentrations of the soluble tumor necrosis factor receptors (sTNFr) I and II rose continuously and significantly during the observation period (p < 0.05). In parallel, a significant rise in nitrate plasma levels was observed (p < 0.05). Moreover, there were highly significant correlations between nitrate and IL-6 serum levels (p < 0.05), nitrate and sTNFr-I (p < 0.05), nitrate and sTNFr-II (p < 0.05), and between IL-6 and IL-10 (p < 0.05), respectively. We conclude that immunotherapy with IL-2 promotes a pro-inflammatory state, parallelled by an increased production of nitric oxide. Although anti-inflammatory responses accompany this process, they are not able to diminish the production of nitric oxide.     

The cytogenetic effects of aflatoxin and gamma-rays on human leukocytes in vitro

Chromosomes from human leukocyte cultures in vitro were treated with γ-rays (200 R), aflatoxin (50 μg/ml, dissolved in dimethyl sulfoxide (DMSO)) and with a combination of both. At the time of treatment (48 h) cells were in all stages of interphase but G₁ cells were evidently predominant. All types of chromosome aberration were observed. Frequencies of chromosome-type aberrations were much higher than those of chromatid type after γ-ray treatment, but these types of chromosome aberration did not differ greatly when the cultures were treated with aflatoxin. Apparently the cytogenetic effect of aflatoxin was delayed longer than was that of irradiation. The present data also suggest the additive effect of γ-rays and aflatoxin in the combined treatment.     

Circulating pro- and anti-inflammatory cytokines in Polish women with gestational diabetes.

In this study we measured serum concentrations of proinflammatory interleukin-6, interleukin-8, and interleukin-18 as well as anti-inflammatory interleukin-10 in 30 pregnant women with normal glucose tolerance, in 32 women with abnormal results of a 50-g glucose challenge test, and in 57 patients with gestational diabetes mellitus. Patients with gestational diabetes had significantly higher IL-6 (median 1.0 [0.7-1.5] vs. 0.7 [0.4-0.8] pg/ml, p=0.001), IL-8 (2.1 [1.1-4.2] pg/ml vs. 0.7 [0.4-0.9] pg/ml, p<0.0001), and IL-18 (249.3 [188.5-318.7] pg/ml vs. 186.7 [139.9-243.9] pg/ml, p=0.005) as well as lower IL-10 levels than healthy pregnant women (0.6 [0.5-1.5] pg/ml vs. 2.9 [1.8-3.2] pg/ml, p<0.0001). After adjusting for glucose, insulin, and BMI values, the differences in IL-8 and IL-18 became insignificant, whereas the differences in IL-6 and IL-10 levels remained highly significant (p<0.0001). The subjects with abnormal glucose challenge test results had higher IL-6 levels (0.9 [0.7-1.3] pg/ml, p=0.005) and similar levels of other cytokines as compared with the women with normal glucose tolerance. Our results suggest an impaired balance between circulating pro- and anti-inflammatory cytokines in patients with gestational diabetes; however, a significant contribution of maternal obesity to the increased levels of IL-8 and IL-18 should be underlined.     

The effect of lipopolysaccharide on anti-inflammatory and pro-inflammatory cytokines production of human amniotic epithelial cells

Intrauterine infection is a major cause of immune imbalance at the maternal-fetal interface, which leads to spontaneous abortion, premature rupture of the fetal membranes, and preterm birth. Human amniotic epithelial cells (hAECs) play a fundamental role in the maintenance of pregnancy. We hypothesize that bacteria influence the immunomodulatory effects of hAECs through stimulation of Toll-like receptors (TLRs). Here, we investigated how lipopolysaccharide (LPS) as a bacterial component affects anti-inflammatory and pro-inflammatory cytokines production of hAECs. Human placentas were obtained from six healthy pregnant women and hAECs were isolated. The phenotypic characteristics of hAECs were determined by flow cytometry. The hAECs (4?×?10⁵ cells/ml) were cultured in the presence or absence of LPS (5?μg/ml). The viability of the cells was assessed and culture supernatants of hAECs were collected after 24, 48 and 72?h of incubation. The levels of transforming growth factor-beta1 (TGF-β1), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-α), interleukin-17?A (IL-17A), and interferon-gamma (IFN-γ) were measured by ELISA. Our data showed that LPS treatment did not affect the viability of hAECs, while had a stimulatory effect on TGF-β1 production of hAECs (p?<?0.001). A significant reduction in IL-4 production of LPS-stimulated hAECs was observed (p?<?0.05). LPS enhanced the production of TNF-α and IL-17?A of hAECs (p?<?0.05–0.0001). The IFN-γ level was only detectable in two culture supernatants of hAECs, and the level was unchanged after stimulation with LPS. Based on these findings, LPS may play a pivotal role in immune imbalance at the feto-maternal interface through affecting anti-inflammatory and pro-inflammatory cytokines production of hAECs.     

Constitutive pro- and anti-inflammatory cytokine and growth factor response to exercise in leukocytes.

Leukocytosis following exercise is a well-described phenomenon of stress/inflammatory activation in healthy humans. We hypothesized that, despite this increase in circulating inflammatory cells, exercise would paradoxically induce expression of both pro- and anti-inflammatory cytokines and growth factors within these cells. To test this hypothesis, 11 healthy adult men, 18-30 yr old, performed a 30-min bout of heavy cycling exercise; blood sampling was at baseline, end-exercise, and 60 min into recovery. The percentage of leukocytes positive for intracellular cytokines and growth factors and mean fluorescence intensity was obtained by flow cytometry. Proinflammatory cytokines (IL-1alpha, IL-2, IFN-gamma, and TNF-alpha), a pleiotropic cytokine (IL-6), and anti-inflammatory cytokines and growth factors [IL-4, IL-10, growth hormone (GH), and IGF-I] were examined. Median fluorescence intensity was not affected by exercise; however, we found a number of significant changes (P < 0.05 by mixed linear model and modified t-test) in the numbers of circulating cells positive for particular mediators. The pattern of expression reflected both pro- and anti-inflammatory functions. In T-helper lymphocytes, TNF-alpha, but also IL-6, and IL-4 were significantly increased. In monocytes, both IFN-gamma and IL-4 increased. B-lymphocytes positive for GH and IGF-I increased significantly. GH-positive granulocytes also significantly increased. Collectively, these observations indicate that exercise primes an array of pro- and anti-inflammatory and growth factor expression within circulating leukocytes, perhaps preparing the organism to effectively respond to a variety of stressors imposed by exercise.     

Modulation of pro- and anti-inflammatory cytokine production in very preterm infants

BACKGROUND: In premature infants, outcome of infection-associated complications is heterogeneous despite advances in antibiotic treatment and diagnosis. Information on the immune response in preterm infants is limited. Immune modulatory strategies require detailed analysis of mediators and their kinetics.OBJECTIVE: To determine the kinetics of IL-1beta, TNFalpha, IL-6, IL-8, IL-10, gammaINF and G-CSF in preterm and term infants in an ex vivo cord blood culture (CBC) endotoxin model. DESIGN AND METHODS: Cord blood of 25 infants was obtained immediately after birth from the fetal side of the placenta and incubated in culture medium (RPMI 1640) in the presence or absence of 500 pg/ml lipopolysaccharide (LPS) for 48h. TNFalpha, IL-1beta, IL-6 and IL-8 were measured by sequential immunometric assay (IMMULITE, DPC Biermann, Germany); IL-10 (Milenia Biotec, Bad Nauheim, Germany), gammaINF (Diaclone, Besancon, France) and G-CSF (R & D Systems, Wiesbaden, Germany) were determined by ELISA in supernatants at 0, 4, 8, 12, 24 and 48h. Infants were stratified into three gestational age groups (< or =32 weeks, 33-36 weeks, > or =37 weeks). Variations between the groups were first analyzed for significance by Kruskal-Wallis test and pairs were compared by Mann-Whitney-U test. Effects of gestational age, leucocyte count, hematocrit and frequency of antenatal steroid exposure were tested by linear regression analysis. To correct a possible impact of variable, WBC count, cytokine levels were adjusted according to individual leucocyte numbers. RESULTS: LPS-stimulated maximum levels of IL-6, IL-1beta,TNFalpha and G-CSF in CBC were significantly lower in very preterm infants compared to more advanced gestational age groups. After adjusting the cytokine levels for 10(5) leucocytes, a significant effect of gestational age on IL-6 and G-CSF production (p<0.05) was detected. A non-significant trend towards reduced cytokine levels was observed following multiple antenatal steroid exposures. IL-10:TNFalpha ratio increased in very preterm neonates when compared with the advanced gestational age, although the increase was not significant. CONCLUSIONS: Pro-inflammatory cytokine activity in CBC correlates with gestational age, whereas IL-10 does not. Although ex vivo synthesis of IL-1beta, TNFalpha, IL-6, G-CSF in CBC depends in part on leucocyte numbers, IL-6 and G-CSF synthesis appeared to be related to immaturity. Non-significant effects of multiple antenatal steroid exposure and increased IL-10:TNFalpha ratio in preterm neonates, observed in a small sample size, warrant further investigation.     

Balance between pro- and anti-inflammatory cytokines in mice treated with Centruroides noxius scorpion venom

CSV consists of a very complex of molecules and demonstrates significant cellular activities capable of stimulating immune functions in vivo. The purpose of this study was to analyze the effects of CSV on sex, weight, route of injection and the balance of pro- and anti-inflammatory cytokines in mice. The susceptibility and route of injection were analyzed by lethal (LD(50)) determination. The effects of CSV were also analyzed in blood from immunized mice using detection by means of antibodies and mediators production. Several functional bioassays were employed: TNF activity was assayed by measuring its cytotoxic activity in L929 cells, and other cytokines were assayed by enzyme-linked immunosorbent assay, whereas nitric oxide levels were detected by Griess colorimetric reactions in sera from BALB/c mice. After injecting subcutaneously, the LD(50) presented an increase of the CSV correlation and similar levels of susceptibility were obtained for female and male from BALB/c mice. Significant differences were observed in the time-course of cytokine levels. The balance of pro- and anti-inflammatory cytokines TNF/IL-10 and IL-6/IL-10 ratios were significantly higher in injected mice group when compared with those obtained for non-injected group. The CSV is poor in antigenic composition and it is difficult to get antibodies specific to neutralizing the lethal factor. The effect of immunization with 0.5 LD(50) of CSV on the balance of pro- and anti-inflammatory cytokines was measured. The maximum levels of TNF and IL-6, IFN-gamma and NO were observed on days 7 and 21 after immunization, respectively. IL-10 levels peaked between days 21 and 28 after immunization with CSV. With respect, to balance of pro- and anti-inflammatory cytokines it was possible to observe that negative correlation between serum levels of IL-6/IL-10 and TNF/IL-10 exists. These ratios may possibly reflect the balance of pro- and anti-inflammatory cytokines in serum, which may by manifested in the inflammatory status during the envenoming processes. In conclusion, an increase in the serum levels of TNF and IL-6 may be a useful marker for scorpion envenomation.     

Biology of recently discovered cytokines: Discerning the pro- and anti-inflammatory properties of interleukin-27

IL-27 is a recently identified heterodimeric cytokine produced in response to microbial and host derived inflammatory cues. Initial studies indicated that IL-27 promotes the generation of Th1 responses required for resistance to intracellular infection and unveiled the molecular mechanisms mediating this effect. However, subsequent work uncovered a role for IL-27 in the suppression of Th1 and Th2 responses. Thus, by discussing its pleotropic functions in the context of infection-induced immunity and by drawing parallels to fellow IL-6/IL-12 family cytokines, this review will attempt to reconcile the pro- and anti-inflammatory effects of IL-27.