Activation of bovine neutrophil functions by interferon-gamma, tumour necrosis factor-alpha, and interleukin-1 alpha

Neutrophils are the first line of defence against microbial infections. They are important in protecting the bovine mammary gland against environmental and pathogenic bacteria such as Escherichia coli and Staphylococcus aureus. Neutrophils in the peripheral blood of healthy cows are functionally a heterogeneous population of cells with markedly varied phagocytic and oxidative activities. Several cytokines have been found to augment leucocyte functions in humans and animal studies. Therefore, the main objective of the present study was to determine if recombinant human interleukin-1 alpha (rhIL-1 alpha), tumour necrosis factor-alpha (rhTNF-alpha), and interferon-gamma (rhIFN-gamma) would potentiate the functional activity of bovine blood neutrophils, thereby providing a possible means to manipulate the resistance of cows to microbial infections.Neutrophils were isolated from the peripheral blood of nine Holstein-Friesian heifers, with a purity of 89%–96% and viability greater than 98%. Aliquots of neutrophils were incubated with five concentrations of rhIL-1 alpha (0.001–10 ng/ml), rhTNF-alpha (0.5–1000 ng/ml), and rhIFN-gamma (0.01-100 ng/ml) at 37°C for 1 h. Then, fluorescein-labelled opsonised zymosan particles were added and the neutrophil suspensions were incubated further for 30 min to evaluate phagocytic activity by fluorescence microscopy. Similarly, unlabelled opsonised zymosan particles and nitroblue tetrazolium (NBT) were added and incubation was carried out for 15 min to evaluate oxidative activity by a spectrophotometric method. Chemotactic activity of cytokine-treated neutrophils for E. coli lipopolysaccharide-activated fetal calf serum was evaluated using a modified Boyden chamber method. The results showed that pretreatment of neutrophils with various concentrations of each cytokine enhanced their phagocytic and NBT reductive activities but reduced chemotactic activity. The phagocytic and NBT reductive activities increased significantly (p0.05) with an increase in the concentration of the cytokines.     

Allele frequencies of polymorphisms of the tumour necrosis factor-alpha, interleukin-10, interferon-gamma and interleukin-2 genes in a North European Caucasoid group from the UK.

Cytokine gene polymorphisms affecting cytokine production may influence rejection and graft-versus-host disease following solid organ and haemopoietic stem cell (HSC) transplantation, respectively. Polymorphisms in the regulatory regions of several cytokine genes have been described; for example, tumour necrosis factor-alpha (TNF-alpha) has a G/A substitution at position -308, interleukin-2 (IL-2) has a T/G substitution at position -330 and interleukin-10 (IL-10) has substitutions at positions -1082(G/A), -819(C/T) and -592(C/A). Microsatellites associated with cytokine production have been detected in the first intron of the IFN-gamma gene and flanking the TNF-alpha gene. In this study, we have genotyped a single panel of healthy Northern European Caucasoids living in the south-east of England for the above-mentioned polymorphisms and compared the results to those published for other populations. A PCR method using sequence-specific primers (SSP) was developed for genotyping the IL-2 polymorphism, and the ABI PRISMtrade mark 310 genetic analyser was used to detect the TNF-alpha and IFN-gamma microsatellites. The allele frequencies of all the studied polymorphisms were consistent with those reported for other UK Caucasoid populations, but differences were observed when compared to other Oriental, African and Caucasoid groups. If these cytokine polymorphisms prove to have functional consequences, then any differences across population groups may have significant clinical relevance in disease and in the outcome of solid organ and HSC transplantation.     

Adrenaline suppression of the macrophage nitric oxide response to lipopolysaccharide is associated with differential regulation of tumour necrosis factor-alpha and interleukin-10

Adrenaline is a catecholamine hormone secreted by the adrenal medulla in response to acute stress. Previous studies have shown that adrenaline suppresses the nitric oxide (NO) response of murine macrophages (M phi s) stimulated in vitro with lipopolysaccharide (LPS). We have now extended these studies to examine the effects of adrenaline on the production of tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10). Our results showed that NO, TNF-alpha and IL-10 were concurrently produced following in vitro LPS (10 micrograms/ml) stimulation of murine peritoneal M phi s. Adrenaline suppressed both NO and TNF-alpha with concomitant up-regulation of the IL-10 response above that seen with LPS alone. In this in vitro model of LPS stimulation we demonstrated that TNF-alpha was required for NO production, as the TNF-alpha neutralizing monoclonal antibody, TN3.19.12, abolished the response; in contrast, IL-10 suppressed NO. In order to determine any functional consequence of adrenaline-mediated IL-10 augmentation on NO production, M phi s were stimulated with LPS and specific neutralizing anti-IL-10 antibodies were added to the cultures. The LPS NO response was suppressed to 43% of the control value by adrenaline (10(-8) M) and an irrelevant control antibody had no effect on the adrenaline-mediated inhibition of NO, but anti-IL-10 treatment restored the NO response to levels similar to those observed with LPS alone. Furthermore, we demonstrated that exogenous TNF-alpha, at a dose range of 1.9-50 ng per ml, also restored the nitrite response to LPS in the presence of adrenaline. Together, the observations that neutralization of IL-10 and addition of TNF-alpha abrogate adrenaline's inhibition of NO, suggest that this hormone suppresses NO partly through up-regulation of IL-10 which, in turn, may suppress TNF-alpha that is required for NO production. Finally, we also observed that the M phi-activating cytokine, interferon-gamma (IFN-gamma), attenuated the inhibitory effect of adrenaline on the LPS NO response.     

Swertia chirayita mediated modulation of interleukin-1beta, interleukin-6, interleukin-10, interferon-gamma, and tumor necrosis factor-alpha in arthritic mice

The effect of aqueous extract of Swertia chirayita stem on the pro- and anti-inflammatory cytokines balance in primary joint synovium of adjuvant-induced arthritic mice has been studied. The level of pro-inflammatory cytokines was found elevated in the joint synovium of arthritic mice in comparison to normal joints. Administration of S. chirayita extract in varying doses through the oral route did not modulate the proinflammatory cytokines on day 2. In contrast, by day 12, a dose dependent (0, 11.86 and 23.72 mg/kg body weight) reduction of tumor necrosis factor-alpha (INF-alpha) interleukin-1beta, (IL-beta) and interferon-gamma, (IFN-gamma) and elevation of Interleukin-10 (IL-10) was observed in the joint homogenates of arthritic mice. Interleukin-6 (IL-6) was not down regulated in joint homogenate of arthritic mice at the dose 11.86 mg/kg but at higher doses (23.72 and 35.58 mg/kg) significant reduction was observed. The aqueous extract was found to possess two polar compounds, amerogentin and mangiferin but was devoid of swerchirin, chiratol, methyl swetianin, and swertanone. Mangiferin has been reported to possess potent anti-inflammatory property and we presume its presence in the aqueous extract of S. chirayita is responsible for reducing TNF-alpha, IL-1beta, IL-6, and IFN-gamma and/or elevating IL-10 in the joint homogenates of arthritic mice on day 12. This study will help in our understanding of the mechanism of anti-inflammatory action of S. chirayita in the light of proinflammatory and anti-inflammatory cytokine balance.     

Eosinophilia, interleukin-5, and tumour necrosis factor-alpha in asthmatic children

BACKGROUND: There are few paediatric studies of the interrelationships between inflammatory markers and asthma severity. We therefore assessed the relationships between eosinophil-associated markers, cytokines, and asthma severity in asthmatic children aged 8-12 years. METHODS: Forty-five children were tested twice, 2 weeks apart. Asthma severity was measured in terms of symptoms, lung function, medication needs, and histamine responsiveness. Peripheral inflammatory markers measured included eosinophil numbers, serum ECP, IL-5, and TNF-alpha and mononuclear cell IL-5, and TNF-alpha production. RESULTS: Histamine responsiveness was correlated with circulating eosinophils (r = 0.56, P = 0.0001) and serum ECP (r = 0.54, P = 0.003). Eosinophilia was increased in children with severe as opposed to mild airway hyperresponsiveness (P = 0.02) and those who lost days at school as opposed to those who did not (P = 0.01). There were no other associations between markers of asthma severity and inflammation. Children taking inhaled corticosteroids had lower serum IL-5 levels than those on beta-agonists +/- cromolyn (mean and 95% CI: 20.5 [11.7-35.7] pg/ml vs 64.3 [26.6-155.4] pg/ml; P = 0.04). Cellular IL-5 production correlated with serum TNF-alpha (r = 0.63, P = 0.0062) and IL-5 (r = -0.59, P = 0.005). CONCLUSION: Serum levels of TNF-alpha and IL-5 were not related to peripheral eosinophilia and asthma severity in these children but were related to their own cellular production ex vivo. This study confirms that eosinophilia is the index of inflammation that is most closely related to the clinical severity of childhood asthma.     

Serum levels of interferon-gamma, tumour necrosis factor-alpha, soluble interleukin-6R and soluble cell activation markers for monitoring response to treatment of leprosy reactions

Identifying pathogen and host-related laboratory parameters are essential for the early diagnosis of leprosy reactions. The present study aimed to clarify the validity of measuring the profiles of serum cytokines [interleukin (IL)-4, IL-6, IL-10, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha], the soluble IL-6 receptor (sIL-6R), soluble T cell (sCD27) and macrophage (neopterin) activation markers and Mycobacterium leprae-specific anti-PGL-I IgM antibodies in relation to the leprosy spectrum and reactions. Serum samples from 131 Indonesian leprosy patients (82 non-reactional leprosy patients and 49 reactional) and 112 healthy controls (HC) from the same endemic region were investigated. Forty-four (89.8%) of the reactional patients had erythema nodosum leprosum (ENL) while only five (10.2%) had reversal reaction (RR). Follow-up serum samples after corticosteroid treatment were also obtained from 17 of the patients with ENL and one with RR. A wide variability in cytokine levels was observed in the patient groups. However, IFN-gamma and sIL-6R were elevated significantly in ENL compared to non-ENL patients. Levels of IFN-gamma, TNF-alpha and sIL-6R declined significantly upon corticosteroid treatment of ENL. Thus, although the present study suggests limited applicability of serial measurement of IFN-gamma, TNF-alpha and sIL-6R in monitoring treatment efficacy of ENL, reactions it recommends a search for a wider panel of more disease-specific markers in future studies.     

T-cell-independent granuloma formation in response to Mycobacterium avium: role of tumour necrosis factor-alpha and interferon-gamma.

We used Mycobacterium avium infection in severe combined immunodeficiency (SCID) mice to examine T-cell-independent mechanisms of inflammatory cell recruitment. SCID mice infected with a virulent strain of M. avium (TMC724) were able to recruit macrophages to sites of mycobacterial replication and formed organized and coherent granulomas in the absence of functional T cells. Phagocyte recruitment was almost totally ablated by neutralization of either tumour necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) in vivo demonstrating that granuloma formation was dependent on the presence of these cytokines. This was concomitant with a reduction in the in situ cytokine mRNA levels otherwise induced in infected mice, for chemokines, pro-inflammatory and regulatory cytokines, including TNF-alpha, IFN-gamma, macrophage inflammatory protein-1 alpha, interleukin-1 beta (IL-1 beta) and IL-10. Furthermore, in vivo treatment of infected mice with anti-asialo GM-1 antisera, which depletes natural killer (NK) cells, prevented recruitment of inflammatory cells. In vitro studies confirmed that M. avium was able to elicit IFN-gamma from SCID spleen in a dose-dependent manner. These data show for the first time that secretion of IFN-gamma from NK cells can mediate a T-cell-independent pathway of granuloma formation and cellular infiltration in response to mycobacteria.     

Reduced in vitro production of interferon-gamma, interleukin-4 and interleukin-12 and increased production of interleukin-6, interleukin-10 and tumour necrosis factor-alpha in systemic lupus erythematosus. Weak correlations of cytokine production with disease activity

Production of cytokines in unstimulated and mitogen-stimulated cultures were evaluated by ELISPOT in 34 SLE patients with low to moderate disease activity and 23 healthy controls. Significantly reduced production of IFN gamma, IL4 and IL12 and significantly increased production of IL6, IL10 and TNF alpha were found in patients with SLE. Regression analysis revealed that production of all six cytokines tended to decrease with increasing disease activity, but negative correlation with SLEDAI was significant (p < 0.05) only for PHA-stimulated IL4, unstimulated and PHA-stimulated IL10 and SAC-stimulated IL6. Negative correlation of stimulated and unstimulated IL6 and TNF alpha production with anti-DNA antibody levels were also significant.     

The effects of dexamethasone and chlorpromazine on tumour necrosis factor-alpha, interleukin-1 beta, interleukin-1 receptor antagonist and interleukin-10 in human volunteers.

Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are pro-inflammatory cytokines that play an important role in severe infections, whereas IL-1 receptor antagonist (IL-1ra) and IL-10 are anti-inflammatory cytokines that counteract their effects. Chlorpromazine and dexamethasone protect mice against lethal endotoxaemia by decreasing circulating concentrations of TNF-alpha and IL-1 beta. We investigated whether administration of chlorpromazine or dexamethasone to human volunteers is able to modulate the lipopolysaccharide (LPS)-stimulated cytokine production capacity in whole blood. Blood samples were taken before and several time-points after medication. Circulating cytokine concentrations were low in all samples. LPS-induced TNF-alpha and IL-1 beta production in whole blood was inhibited by dexamethasone treatment, while chlorpromazine had no effect. When peripheral blood mononuclear cells were stimulated in vitro with LPS, the addition of chlorpromazine (1-100 ng/ml) had no modulatory action on TNF-alpha, IL-1 beta, IL-1ra or IL-10 synthesis. The chlorpromazine concentrations measured in circulation of volunteers were eight to 40 times lower than the concentrations shown to be effective in mice. In conclusion, chlorpromazine inhibits TNF-alpha and IL-1 beta production in mice at concentrations that cannot be reached in humans, thus precluding its usage in clinical anti-cytokine strategies. In contrast, dexamethasone is an effective inhibitor of pro-inflammatory cytokine production.     

Detection of in vitro interferon-gamma and serum tumour necrosis factor-alpha in multidrug-resistant tuberculosis patients

Multidrug-resistant tuberculosis (MDR-TB) is known as having a poor prognosis with a weak response to therapy and very high death rates. The aim of this work was to assess the immune response to the RD1-encoded antigen ESAT-6 of Mycobacterium tuberculosis in MDR-TB patients and compare to non-resistant (NR) TB patients and healthy controls (HC). Evaluation of interferon (IFN)-gamma production showed that, although 55% of the MDR patients were responsive to ESAT-6, they produced lower IFN-gamma levels (553 +/- 11 pg/ml) when compared to NR-TB (1179 +/- 163 pg/ml; P < 0.05) but not to controls (412 +/- 65.7 pg/ml). Differences in the response to ESAT-6 and to its overlapping peptides mixture were also significant between MDR versus treated pulmonary NR-TB. Furthermore, a very low rate of response to PPD (23.5%) and to Ag85B (33.3%) was noted in MDR-TB patients as compared to the other groups. To determine the inflammatory response in patients' groups, detection of tumour necrosis factor (TNF)-alpha was assessed in their sera before and during chemotherapy. Mean TNF-alpha levels in MDR-TB (43.8 +/- 9 pg/ml) paralleled those found in treated pulmonary, and it was significantly different (P < 0.05) from the values found in untreated NR and HC. Interestingly, secretion of IFN-gamma and TNF-alpha were predominant in MDR patients who presented with bilateral pulmonary lesions and lung cavitation. The present data indicate that the overall immune response to mycobacterial antigens is decreased in resistant TB and the major role inflammatory cytokines may play in perpetuating pulmonary tissue damage.     

The roles of tumour necrosis factor-alpha, interleukin-1 and interleukin-12 in murine cytomegalovirus infection.

The swine is a useful model for immunobiological studies as it has a highly heterogeneous lymphocyte pool, containing several subsets not easily accessible in humans and rodents. In particular, the CD8-positive (CD8+) cells contain a variety of lymphocyte subsets, such as alpha beta-T cells, gamma delta-T cells, CD4 CD8 double-positive (DP) cells and natural killer (NK) cells. In order to define these subsets further, we have selected four monoclonal antibodies (mAb) with differential reactivity on CD8+ cells. Thus, mAb CD8.1 (PPT20) bound to CD8hi and CD8lo subpopulations in a similar way to the conventional anti-CD8. The mAb CD8.2 (PPT21), though binding to all of the CD8+ cells, reacted preferably with CD8hi. Two other mAb, CD8.3 (PPT22) and CD8.4 (PPT23), were specific for CD8hi alpha beta-T-cell subpopulation. These results, complemented by immunoprecipitation, co-modulation and enzyme-linked immunosorbent assay experiments, suggest that CD8.1 and CD8.2 react putatively with the CD8 alpha-chain and CD8.3 and CD8.4 with the CD8 beta-chain. Tissue distribution studies revealed that CD8+ thymocytes and peripheral CD8hi alpha beta-T cells expressed both putative CD8 alpha- and beta-chains while peripheral CD4+ CD8+ alpha beta-T cells, CD8lo gamma delta-T cells and NK cells expressed only putative CD8 alpha-chain. Functional studies indicated that the CD8hi alpha beta-T and CD8lo gamma delta-T cells were effector cells in the CD3-redirected cytotoxicity.     

Augmentation of the human monocyte/macrophage chemiluminescence response during short-term exposure to interferon-gamma and tumour necrosis factor-alpha.

The effects of short-term (30 min) pre-incubation of human monocytes and macrophages (3-day cultured monocytes) with leucocyte-derived human interferon-gamma (IFN-gamma) and recombinant human tumour necrosis factor-alpha (rTNF-alpha) were examined. Pre-incubation of either monocytes or macrophages with rTNF-alpha or IFN-gamma (100 U/5 x 10(5) cells) augmented their respiratory burst to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), measured by the luminol- and lucigenin-dependent chemiluminescence assay. In addition, both cell types showed a burst of respiratory activity in the presence of rTNF-alpha or IFN-gamma only. The effects of IFN-gamma were removed by adsorption with an anti-IFN-gamma monoclonal antibody and those of rTNF-alpha were abolished by heating at 100 degrees C, or by the addition of anti-TNF-alpha monoclonal antibody. The results demonstrate that both IFN-gamma and rTNF-alpha are stimulators of monocytes and macrophages, and rapidly alter the capacity of the cells to respond to fMLP, which binds to cell surface receptors.     

Human neutrophils activated by interferon-gamma and tumour necrosis factor-alpha kill Entamoeba histolytica trophozoites in vitro

The interaction between cytokine-activated human neutrophils and Entamoeba histolytica trophozoites was studied as well as the mechanism(s) involved. Treatment of neutrophils with rIFN-gamma alone allowed them to kill 30% of E. histolytica trophozoites; however, rIFN-gamma and rTNF-alpha pretreatments in combination increased neutrophil killing to 70%. In the absence of direct contact between neutrophils and amebae, rIFN-gamma-treated neutrophils were shown to kill 70% of amebae, and rIFN-gamma- and rTNF-alpha-treated neutrophils killed 97% of amebae. Neutrophil enhancement of amebicidal activity following cytokine treatments was correlated with increased neutrophil resistance to amebic contact-dependent killing and was shown to be 73% H2O2 dependent.     

Predicting death from tumour necrosis factor-alpha and interleukin-6 in 80-year-old people

Ageing is associated with low-grade inflammation and markers such as IL-6 possess prognostic value. Tumour necrosis-alpha (TNF-alpha) initiates the inflammatory cascade and has been linked to several age-associated disorders. It remains, however, unknown if TNF-alpha is associated with mortality in old populations. The aim of the present study was to investigate if serum levels of TNF-alpha were associated with all-cause mortality independently of interleukin (IL)-6 in a prospective study of 333 relatively healthy 80-year-old people. A Cox regression model was used to explore effects of TNF-alpha and IL-6 on survival in the following 6 years. A total of 133 participants died during this follow-up period. TNF-alpha was associated with mortality in men, but not in women, whereas low-grade elevations in IL-6 were associated strongly with mortality in both sexes. TNF-alpha explained only 7% of the variability in IL-6 and effects of the two cytokines were independent of each other as well as of other traditional risk factors for death [smoking, blood pressure, physical exercise, total cholesterol, co-morbidity, body mass index (BMI) and intake of anti-inflammatory drugs]. These findings indicate that at least in old populations chronic elevated levels of TNF-alpha and IL-6 have different biological functions that trigger age-associated pathology and cause mortality.     

A ceramide-1-phosphate analogue, PCERA-1, simultaneously suppresses tumour necrosis factor-alpha and induces interleukin-10 production in activated macrophages

Tight regulation of the production of the key pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a synthetic phospholipid, named hereafter phospho-ceramide analogue-1 (PCERA-1), was previously found to suppress lipopolysaccharide (LPS)-induced TNF-alpha blood levels. We therefore investigated the in vitro anti-inflammatory effects of PCERA-1. Here, we show that extracellular PCERA-1 potently suppresses production of the pro-inflammatory cytokine TNF-alpha in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Specificity is demonstrated by the inability of the phospholipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF-alpha suppression and IL-10 induction by PCERA-1 were observed in macrophages when activated by Toll-like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA-1 does not block activation of nuclear factor (NF)-kappaB and mitogen-activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the anti-inflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF-alpha suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.     

Evaluation of nonspecific immunity and plasma levels of interferon-gamma, interleukin-6 and tumor necrosis factor-alpha in preeclampsia.

In this study we evaluated the maternal cell-mediated immune aspects of preeclampsia in terms of phagocytosis and killing of monocytes and polymorphonuclear cells. To evaluate the contribution of cytokines (Cks) in the pathophysiology of preeclampsia, we investigated the plasma levels of interferon-gamma (IFN-gamma), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), respectively, by an enzyme-linked immunosorbent assay. Data showed that phagocytic and killing activities of monocytes were depressed in preeclamptic and normal pregnancies. At the same time, IFN-gamma plasma levels were undectable in both groups. Conversely, we detected significant levels of TNF-alpha in plasma from preeclamptic and normal pregnancies. Moreover, since in three preeclamptic patients the onset of severe preeclampsia was associated with a sharp increased of TNF-alpha plasma levels, we suggest that an increased production of this CK may be implicated in the pathophysiology of preeclampsia.     

Induction and suppression of cytokine release (tumour necrosis factor-alpha; interleukin-6, interleukin-1 beta) by Escherichia coli pathogenicity factors (adhesions, alpha-haemolysin).

Escherichia coli bacteria expressing mannose-resistant fimbriae/haemagglutination induced the production of substantial amounts of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) from a peripheral human lymphocyte, monocyte, basophil (LMB) cell suspension. In this regard, E. coli bacteria with S-mannose-resistant fimbriae/haemagglutination were the most potent inducers of IL-6 and TNF-alpha secretion, followed by the E. coli strain with P-mannose-resistant fimbriae/haemagglutination. The E. coli alpha-haemolysin did not stimulate cytokine release from human LMB. In fact, this toxin, at non-toxic concentrations, depressed the spontaneous as well as the E. coli-induced production of TNF-alpha, IL-6, IL-1 beta. Our results indicate that two mechanisms may contribute to the severity of E. coli infection: (a) stimulation of cytokine release by type-specific fimbriae/haemagglutination properties and (b) depression of immune response by the E. coli alpha-haemolysin.