Quantifying mating success of territorial males and sneakers in a bower-building cichlid fish.

The strategies and traits males evolve to mate with females are incredible in their diversity. Theory on the evolution of secondary sexual characters suggests that evolving any costly trait or strategy will pay off and stabilise in the population if it is advantageous compared to the alternative less costly strategy, but quantifying the relative success of the two can be difficult. In Lake Malawi, Africa, there are >200 species of cichlid fish in which the males form leks and spend several weeks per year building sand-castle "bowers" several times their size. We tested the idea that a less costly "sneaking" strategy could be successful by quantifying the mating success of bower-holding versus non-bower-holding males. We PIT-tagged every fish in a semi-natural experimental set-up and placed tag-readers on the side of bowers to determine which fish held a bower. We then genotyped the eggs removed from females' mouths to assign paternity of each egg. Broods were fathered by up to 3 different males. Although paternity was mostly assigned to males that held a bower, a small number of males who did not own a bower were more successful than some of those that did, indicating a role for an alternative strategy in these bower builders.

Community Genetics Reveal Elevated Levels of Sympatric Gene Flow among Morphologically Similar but Not among Morphologically Dissimilar Species of Lake Victoria Cichlid Fish.

We examined genetic structure among five species of Lake Victoria haplochromine cichlids in four island communities, using a full factorial sampling design that compared genetic differentiation between pairs of species and populations of varying morphological similarity and geographical proximity. We found that allopatric conspecific populations were on average significantly more strongly differentiated than sympatric heterospecific populations of morphologically similar species. Allopatric heterospecific populations of morphologically dissimilar species were most differentiated. Our work demonstrates that phenotypic divergence can be maintained and perhaps even evolve in sympatry despite considerable gene flow between species. Conversely, phenotypic resemblance among conspecific populations can be maintained despite geographical isolation. Additionally we show that anthropogenically increased hybridization does not affect all sympatric species evenly but predominantly affects morphologically similar and closely related species. This has important implications for the evolution of reproductive isolation between species These findings are also consistent with the hypothesis of speciation reversal due to weakening of divergent selection and reproductive isolation as a consequence of habitat homogenization and offers an evolutionary mechanistic explanation for the observation that species poor assemblages in turbid areas of the lake are characterized by just one or two species in each of a few morphologically distinct genera.

Importance of time and place: patterns in abundance of Symbiodinium clades A and B in the tropical sea anemone Condylactis gigantea.

The capacity of some corals and other cnidarians to form symbioses with multiple algae (Symbiodinium) is a candidate route by which these symbioses tolerate variable environmental conditions. On Bermuda, the coral reef dwelling anemone Condylactis gigantea bears Symbiodinium of clades A and B. At thermally variable inshore and nearshore sites, clade A predominates (as sole symbiont or in mixed infection with clade B), whereas animals at offshore sites with more uniform temperatures bear only clade B or mixed infections. Individual animals at one nearshore site monitored over a year by sampling tentacles showed increased prevalence of clade A in March-November, when sea waters were warm (average 26 degrees C), and increased clade B in November-March when cool waters prevailed (average 18.5 degrees C). In laboratory analyses of excised tentacles, the symbiosis with clade B, but not clade A, bleached at elevated temperature (32 degrees C), suggesting that thermal tolerance may contribute to the higher prevalence of clade A at inshore/nearshore sites and in the summer. The temporal changes in the algal complement were not accompanied by bleaching, and Symbiodinium density fluctuated in hosts with stable Symbiodinium composition but not in hosts with variable composition. This suggests that changes in the relative abundance of Symbiodinium clades do not require bleaching and may even protect the symbiosis from large fluctuations in algal density.

Indirect mate choice, direct mate choice and species recognition in a bower-building cichlid fish lek.

Sexual selection arising through female mate choice typically favours males with larger, brighter and louder signals. A critical challenge in sexual selection research is to determine the degree to which this pattern results from direct mate choice, where females select individual males based on variation in signalling traits, or indirect mate choice, where male competition governs access to reproductively active females. We investigated female mate choice in a lekking Lake Malawi cichlid fish, Hemitilapia oxyrhynchus, in which males build and aggressively defend sand 'bowers'. Similar to previous studies, we found that male reproductive success was positively associated with bower height and centrality on the lek. However, this pattern resulted from males holding these territories encountering more females, and thus their greater success was due to indirect mate choice. Following initial male courtship, an increase in the relative mating success of some males was observed, but this relative increase was unrelated to bower size or position. Crucially, experimentally manipulating bowers to resemble those of a co-occurring species had no appreciable effect on direct choice by females or male spawning success. Together, these results suggest indirect mate choice is the dominant force determining male-mating success in this species, and that bowers are not signals used in direct mate choice by females. We propose that, in this species, bowers have a primary function in intraspecific male competition, with the most competitive males maintaining larger and more central bowers that are favoured by sexual selection due to higher female encounter rates.

Evolution of MHC class IIB in the genome of wild and ornamental guppies, Poecilia reticulata.

This is the first study to quantify genomic sequence variation of the major histocompatibility complex (MHC) in wild and ornamental guppies, Poecilia reticulata. We sequenced 196-219 bp of exon 2 MHC class IIB (DAB) in 56 wild Trinidadian guppies and 14 ornamental strain guppies. Each of two natural populations possessed high allelic richness (15-16 alleles), whereas only three or fewer DAB alleles were amplified from ornamental guppies. The disparity in allelic richness between wild and ornamental fish cannot be fully explained by fixation of alleles by inbreeding, nor by the presence of non-amplified sequences (ie null alleles). Rather, we suggest that the same allele is fixed at duplicated MHC DAB loci owing to gene conversion. Alternatively, the number of loci in the ornamental strains has contracted during >100 generations in captivity, a hypothesis consistent with the accordion model of MHC evolution. We furthermore analysed the substitution patterns by making pairwise comparisons of sequence variation at the putative peptide binding region (PBR). The rate of non-synonymous substitutions (dN) only marginally exceeded synonymous substitutions (dS) in PBR codons. Highly diverged sequences showed no evidence for diversifying selection, possibly because synonymous substitutions have accumulated since their divergence. Also, the substitution pattern of similar alleles did not show evidence for diversifying selection, plausibly because advantageous non-synonymous substitutions have not yet accumulated. Intermediately diverged sequences showed the highest relative rate of non-synonymous substitutions, with dN/dS>14 in some pairwise comparisons. Consequently, a curvilinear relationship was observed between the dN/dS ratio and the level of sequence divergence.

Targets of glucocorticoid action on TNF-alpha release by macrophages.

Glucocorticoid drugs affect virtually every cell type involved in inflammatory response, to some degree. Macrophage/monocytes (Mphi) are particularly sensitive, and glucocorticoids suppress release of most known Mphi inflammatory mediators, including TNF-alpha. In the case of TNF-alpha, several levels of regulation are already characterised and ongoing research hints at further glucocorticoid targets. The relative importance of transcriptional and post-transcriptional regulation is lineage-dependent and may also change during the course of Mphi differentiation. In human monocytic cell lines, glucocorticoids primarily suppress transcriptional activation through adjacent promoter binding sites for NF-kappaB transcription factor complexes and for complexes of c-Jun with activating transcription factor-2 (ATF-2). The goal of glucocorticoid research in inflammation is to develop drugs with the anti-inflammatory potential of glucocorticoids, but without the systemic toxicity. Each of the multiple targets for glucocorticoid action presents an opportunity for anti-inflammatory drug development. However, none of the known targets is unique to Mphi, and no single pathway is preeminent in all situations. Research is now directed at characterising targets and regulating them without systemic activation of the glucocorticoid receptor.

Glucocorticoids suppress tumor necrosis factor-alpha expression by human monocytic THP-1 cells by suppressing transactivation through adjacent NF-kappa B and c-Jun-activating transcription factor-2 binding sites in the promoter.

Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis by activated monocyte/macrophages, contributing to an anti-inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-alpha transcription. Site-directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-alpha promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-alpha 3'-untranslated region. We conclude that glucocorticoids suppress LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding sites in the -106 to -88 bp region of the TNF-alpha promoter.

Effects of stimulus and cell type on the expression of the -308 tumour necrosis factor promoter polymorphism.

The authors have previously demonstrated that the tumour necrosis factor (TNF) -308 G/A polymorphism affects the binding of transcription factors. In transient transfection assays in PMA stimulated U937 monocytes and Jurkat T cells, the A-containing TNF2 promoter has a 2-3-fold greater transcriptional activity than the TNF1 promoter in the presence of the TNF 3'UTR. In this study it was found that a difference in TNF1 and TNF2 promoter activities was only observed in U937 and Jurkat cells, and not in Raji (B cell line), HeLa (epithelial carcinoma cell line), HepG2 (hepatoma cell line) or THP-1 (monocyte), suggesting cell-type specific transcription factors or modifications may be involved in the formation of the -308 protein/DNA complex. Physiological stimulators, TNF and interferon gamma (IFN-gamma) did not cause differential promoter activity between TNF1 and TNF2, but LPS did with only the TNF2 promoter/3'UTR construct being significantly responsive to lipopolysaccharide (LPS) in U937 cells. In U937 cells, the -308 polymorphism affected transcription following differentiation by phorbol myristate acetate (PMA), retinoic acid, PMA plus LPS and PMA plus retinoic acid with an increase in nuclear factor binding to both TNF1 and TNF2 in the -323 to -285 region being observed. The greatest difference between TNF2 and TNF1 promoter activities (5-fold) was observed following PMA plus retinoic acid treatment of transfected U937 cells for 48h. During this time, U937 differentiated into cells with a macrophage-like morphology. An understanding of the cell type and stimuli specific requirements for differential expression of the -308 polymorphism may help elucidate the role the TNF -308 polymorphism plays in diseases where elevated TNF levels are thought to be important.

Altered leucocyte trafficking and suppressed tumour necrosis factor alpha release from peripheral blood monocytes after intra-articular glucocorticoid treatment.

OBJECTIVES: A generalised transient improvement may follow intra-articular administration of glucocorticoids to patients with inflammatory arthropathy. This may represent a systemic anti-inflammatory effect of glucocorticoid released from the joint, mediated through processes such as altered leucocyte trafficking or suppressed release of pro-inflammatory cytokines. Patients, who had received intra-articular injections of glucocorticoids were therefore studied for evidence of these two systemic effects. METHODS: Patients with rheumatoid arthritis were studied. Peripheral blood leucocyte counts, tumour necrosis factor alpha (TNF alpha) release by peripheral blood monocytes, blood cortisol concentrations, and blood methylprednisolone concentration were measured for 96 hours after intra-articular injection of methylprednisolone acetate. RESULTS: Measurable concentrations of methylprednisolone were present in blood for up to 96 hours after injection. Significant suppression of the hypothalamic-pituitary-adrenal axis persisted throughout this time. Altered monocyte and lymphocyte trafficking, as evidenced by peripheral blood monocytopenia and lymphopenia, was apparent by four hours after injection and resolved in concordance with the elimination of methylprednisolone. Granulocytosis was observed at 24 and 48 hours. Release of TNF alpha by endotoxin stimulated peripheral blood monocytes was suppressed at four hours and thereafter. Suppression was maximal at eight hours and was largely reversed by the glucocorticoid antagonist, mifepristone. CONCLUSIONS: After intra-articular injection of methylprednisolone, blood concentrations of glucocorticoid are sufficient to suppress monocyte TNF alpha release for at least four days and to transiently alter leucocyte trafficking. These effects help to explain the transient systemic response to intra-articular glucocorticoids. Suppression of TNF alpha is principally a direct glucocorticoid effect, rather than a consequence of other methylprednisolone induced changes to blood composition.

Dexamethasone suppresses release of soluble TNF receptors by human monocytes concurrently with TNF-alpha suppression.

Glucocorticoids suppress many monocyte functions, including endotoxin-stimulated release of TNF-alpha. Monocytes also release soluble receptors for TNF (sTNF-R), which can modulate TNF bioactivity. We therefore examined the effects of the glucocorticoid, dexamethasone, on the release of soluble forms of the 55 kDa and 75 kDa receptors for TNF (sTNF-R55 and sTNF-R75) by human monocytes and the human monocytic Mono Mac 6 cell line. Peripheral blood mononuclear cells (PBMC) spontaneously released 406 +/- 181 pg/10(6) cells of sTNF-R75 over 18 h in culture and Mono Mac 6 cells released 554 +/- 29 pg/10(6) cells. Lipopolysaccharide (LPS) exposure increased release of sTNF-R75 by 54 and 217%, respectively. Dexamethasone suppressed both spontaneous and LPS-stimulated release. The effect of dexamethasone was concentration dependent. At 1 mumol/L, dexamethasone suppressed the LPS-stimulated release of sTNF-R75 by 86% in PBMC and by 40% in Mono Mac 6 cells. Neither PBMC nor Mono Mac 6 cells released measurable amounts of sTNF-R55, but spontaneous release of sTNF-R55 from purified human monocytes (55 +/- 2 pg/10(6) cells over 18 h) was reduced by 45% in the presence of dexamethasone. Dexamethasone reduced bioactive TNF in PBMC cultures, as well as immunoassayable TNF-alpha, which indicates that suppression of TNF-alpha release was biologically more important than suppressed release of soluble inhibitors. Similar concurrent suppression of IL-1 beta and IL-1ra release occurred in PBMC and Mono Mac 6 cultures exposed to dexamethasone.

Suppression of human monocyte tumour necrosis factor-alpha release by glucocorticoid therapy: relationship to systemic monocytopaenia and cortisol suppression.

AIMS: Glucocorticoids suppress the release of tumour necrosis factor-alpha (TNF-alpha) by macrophages in vitro and cause monocytopaenia in vivo. These actions may contribute to anti-inflammatory and immunosuppressant effects. We therefore examined relationships between prednisolone concentration, suppression of monocyte TNF-alpha release, monocytopaenia and suppression of total cortisol concentration in healthy volunteers treated with a single dose (1.5 mg kg-1) of the glucocorticoid, prednisolone. METHODS: Monocyte numbers, total cortisol concentration and prednisolone concentration were measured in blood samples collected over 48 h after the dose. Plasma from these samples was also tested for its capacity to suppress lipopolysaccharide-induced TNF-alpha release from monocytes in autologous whole blood cultures. RESULTS: At 4 h after the dose, monocyte numbers in peripheral blood had fallen to a mean of 18% of the pre-dose level whilst plasma total cortisol had fallen to 9% of the pre-dose concentration. Monocyte numbers recovered in concordance with elimination of prednisolone and there was a significant relative monocytosis at 24 h. The recovery of plasma cortisol was delayed in comparison, with cortisol remaining significantly suppressed at 24 h. Plasma samples taken at 2 h after the dose (corresponding to peak plasma prednisolone concentration) suppressed the lipopolysaccharide-stimulated production of TNF-alpha by autologous blood monocytes to 27% of pre-dose control. Plasma collected at intervals over the 48 h from dosing also suppressed monocyte TNF-alpha release in relation to the prednisolone concentration therein. Suppression was largely reversed by the glucocorticoid antagonist, mifepristone. A similar relationship between prednisolone concentration and TNF-alpha suppression was observed when prednisolone was added to blood samples collected from the volunteers when they were drug-free. CONCLUSIONS: Blood concentration of prednisolone achieved after a dose of 1.5 mg kg-1 are sufficient to suppress monocyte TNF-alpha release and cause a biphasic change in peripheral blood monocyte numbers. Suppression of TNF-alpha is principally a direct glucocorticoid effect, rather than a consequence of other prednisolone-induced changes to blood composition.

Glucocorticoid modulation of human monocyte/macrophage function: control of TNF-alpha secretion.

Glucocorticoids suppress many functions in activated monocyte/macrophages, including the release of TNF-alpha. This is likely to contribute to the efficacy of glucocorticoids in some inflammatory diseases, such as rheumatoid arthritis, where TNF-alpha contributes to pathogenesis. Glucocorticoids suppress the activity of reporters which include TNF-alpha promoter regions and modify the activity of NF-kappa B family transcription factors in activated human monocytic cell lines, suggesting effects of glucocorticoids on TNF-alpha gene transcription. In addition, glucocorticoids have been reported to antagonise the enhanced translational efficiency of TNF-alpha mRNA which occurs at least after stimulation of murine monocytic cells. It is likely, therefore, that glucocorticoids act at several points in stimulated monocyte/ macrophages to reduce TNF-alpha secretion. Understanding glucocorticoid control of TNF-alpha secretion may explain some of the variability in response to GC in inflammatory diseases and may reveal means of inducing glucocorticoid-like anti-inflammatory effects in monocyte/macrophages without exposing other tissues to the adverse effects of glucocorticoids.

Dexamethasone antagonizes IL-4 and IL-10-induced release of IL-1RA by monocytes but augments IL-4-, IL-10-, and TGF-beta-induced suppression of TNF-alpha release.

The activities of monocyte-derived tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta are potentially modified by IL-1RA and soluble receptors for TNF (sTNF-R), which are themselves monocyte products. IL-4, IL-10, TGF-beta, and glucocorticoids (GC) all suppress the lipopolysaccharide (LPS)-stimulated release of TNF-alpha and IL-1beta but vary in their effects on IL-1RA and sTNF-R. This raises the prospect of interactions between the cytokines and glucocorticoids, which may be antagonistic or additive on IL-1 and TNF activity. We, therefore, studied the interactions of the GC dexamethasone (Dex) with IL-4, IL-10, and transforming growth factor (TGF)-beta on the release of TNF-alpha and IL-1RA by human monocytes and the monocytic THP-1 cell line. Low concentration of Dex (10(-8)-10(-7)M) acted additively with low concentrations of IL-4 (0.01-1 ng/ml), IL-10 (0.01-0.1 U/ml), or TGF-beta (0.01-1 ng/ml) to profoundly suppress LPS-stimulated release of TNF-alpha by whole blood and, to a lesser degree, THP-1 cells. Dex also suppressed spontaneous release of IL-1RA from PBMC and THP-1 cells, whereas IL-4 and IL-10, but not TGF-beta, stimulated release. Dex antagonized the enhanced release in IL-4 and IL-10-stimulated cultures. The capacity to stimulate release of IL-1RA may contribute to the anti-inflammatory potential of IL-4 and IL-10 in monocyte/macrophage-mediated disease. GC, therefore, do not uniquely enhance the suppressive functions of IL-4 and IL-10 on monokine activity. The therapeutic benefit of combinations of GC and IL-4, IL-10 or TGF-beta in disease may depend on the roles of the individual monokines and antagonists in pathogenesis.

IL-4, IL-10 and IFN-gamma have distinct, but interacting, effects on differentiation-induced changes in TNF-alpha and TNF receptor release by cultured human monocytes.

Monocytes cultured in vitro differentiate to a macrophage-like phenotype and undergo functional changes, including reduced capacity for release of TNF-alpha and the soluble p55 receptor for TNF (sTNF-R55) but enhanced capacity for release of the soluble p75 receptor (sTNF-R75). The cytokines IL-4 and IL-10 act on monocytes to suppress the release of pro-inflammatory cytokines, including TNF-alpha, and to influence the release of sTNF-R. We therefore investigated the influence of differentiation over 15 days in vitro on the spontaneous and LPS- and IFN-gamma-induced release of TNF-alpha and sTNF-R from human monocytes and examined the actions of IL-4 and IL-10 on these. Unstimulated monocytes did not release TNF-alpha at any stage but released progressively larger amounts of sTNF-R75 with time. LPS-stimulated release of TNF-alpha declined substantially after the first day and was consistently suppressed by IL-10 and IL-4 but increased by IFN-gamma. Monocytes cultured with IL-10 released more sTNF-R75 at all times and expressed more mRNA for TNF-R75 at day 8. LPS stimulation consistently enhanced both spontaneous and IL-10-augmented release of sTNF-R75, whilst IFN-gamma co-stimulation consistently suppressed them. The influence of IL-4 on sTNF-R75 release, however, depended qualitatively on both the length of time in culture and on conditions of stimulation. The effects of LPS and IFN-gamma on TNF-alpha and sTNF-R75 release were progressively lost with increasing time in culture in the presence of IL-4. sTNF-R55 was not detectable after the first day of culture under any of these conditions. IL-4, IL-10 and IFN-gamma therefore have distinct, but interacting, effects on the balance between TNF-alpha and sTNF-R75 release by maturing monocytes. These interactions may be relevant to the pathogenesis or treatment of TNF-alpha-mediated diseases, where sTNF-R may act to neutralize or stabilise TNF, thereby modifying biological activity.

Tumor necrosis factor alpha and interleukin-1 alpha stimulate late shedding of p75 TNF receptors but not p55 TNF receptors from human monocytes.

Soluble receptors for TNF (sTNF-R) are present at elevated concentrations in the synovial fluid of patients with rheumatoid arthritis. They are presumably released by cells of the synovial membrane, including the monocyte-derived synovial macrophages. Cytokines from the synovium, including IL-1 and TNF-alpha, may stimulate release. We therefore examined the release of sTNF-R from monocytes exposed to IL-1 and TNF-alpha. Elutriator-purified human blood monocytes spontaneously released both the p75 and the p55 sTNF-R (1011 +/- 199 and 177 +/- 20 pg/10(6) cells, respectively, mean +/- SEM) during 48 h of in vitro culture. TNF-alpha and IL-1 alpha induced time- and concentration-dependent increases in the release of sTNF-R75 from monocytes, but neither had a measurable effect on the release of sTNF-R55. The release of sTNF-R75 was inhibited by cycloheximide. Neither lymphocytes nor polymorphonuclear leukocytes (PMN) released measurable sTNF-R spontaneously or in response to stimulation with IL-1 alpha, but TNF-alpha stimulated the release of small amounts of sTNF-R75 by PMN. The timing, cycloheximide sensitivity, and selectivity of stimulated release of TNF-R75 by monocytes are consistent with previous observations on other cell types of late (8-20 h) increased synthesis and turnover of cell surface TNF-R75, but not TNF-R55, after stimulation with TNF-alpha or IL-1. These observations help to explain why elevated levels of sTNF-R in synovial fluid coexist with enhanced expression of cell surface TNF-R on synovial macrophages in rheumatoid arthritis.

A system for assessment of monokine gene expression using human whole blood.

Monocyte derived cytokines (monokines) are important mediators in inflammatory diseases and cancer. Control of monokine expression is also a major therapeutic target in autoimmune inflammation. Whole blood cultures permit examination of monokine expression under conditions which emulate the in-vivo environment whilst avoiding many of the artefacts associated with monocyte separation and culture. Here we describe a system for measuring interleukin-1 beta, interleukin-1 alpha, interleukin-6 and tumour necrosis factor-alpha mRNA in stimulated human whole blood ex-vivo, which can be applied to specimens from treated patients. Oligodeoxyribonucleotide probes are designed to allow standardisation of hybridisation and washing procedures. Washing and reprobing of membranes in appropriate sequence permits measurement of each monokine mRNA and mRNA for glyceraldehyde-3-phosphate dehydrogenase in only 7 ml of lipopolysaccharide-stimulated human blood. The method has been used successfully in studies of dexamethasone and methotrexate action on lipopolysaccharide stimulated IL-beta gene expression.

Two inhibitors of pro-inflammatory cytokine release, interleukin-10 and interleukin-4, have contrasting effects on release of soluble p75 tumor necrosis factor receptor by cultured monocytes.

The biological activity of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha depends on the level of TNF-alpha itself, the expression of the p55 and p75 cell surface receptors for TNF on target cells and the concentrations of the natural inhibitors of TNF-alpha, the soluble p55 and p75 TNF receptors (TNF-R). Interleukin (IL)-10 and IL-4 are known to inhibit TNF-alpha production by monocytes. We, therefore, investigated the effects of IL-10 and IL-4 on the cell surface expression and release of TNF-R by human monocytes to determine whether these cytokines also indirectly modulated the biological activity of TNF-alpha. Exposure to IL-10 (1-10 U/ml) for 24 or 48 h increased soluble p75 TNF-R expression and concomitantly reduced surface expression of p75 TNF-R. Further, IL-1 alpha-stimulated production of TNF-alpha was diminished by IL-10 and only a small proportion of this TNF-alpha was bioactive, consistent with increased production of inhibitory soluble TNF-R. IL-10 also induced down-regulation of surface p55 TNF-R on monocytes, and increased release of soluble p55 TNF-R. However, the expression of soluble p55 TNF-R was much lower than soluble p75 TNF-R, indicating that it contributed less importantly to neutralization of TNF-alpha under these conditions. Like IL-10, IL-4 supressed the release of TNF-alpha by monocytes. In contrast to IL-10, however, IL-4 (0.1-10 ng/ml) supressed the release of soluble p75 TNF-R from monocytes in a dose-dependent manner. Release of soluble p55 TNF-R was also supressed by IL-4. IL-10, therefore, reduces the pro-inflammatory potential of TNF in three ways: by down-regulating surface TNF-R expression whilst increasing production of soluble TNF-R and inhibiting the release of TNF-alpha itself. This suggests that IL-10 may be useful in the treatment of diseases where overexpression of TNF-alpha occurs.