Validation of a new highly standardised, lab-independent whole-blood leukocyte function assay for clinical trials (ILCS).

Physical stress induced in healthy volunteers by the Loughborough intermittent shuttle test (LIST) was used to validate a newly developed whole-blood cell culture system (Instant leukocyte culture system (ILCS). Exercise induced immune modulation was investigated through measurement of cytokine levels after activating leukocytes in peripheral blood ex vivo using the physiologic stimulant lipopolysaccharide (LPS). LPS induced production of three different cytokines, interferon gamma (IFNgamma), interleukin-6 (IL-6), and interleukin-10 (IL-10). IFNgamma levels were significantly decreased (P = 0.02 and P = 0.001 ) and IL-10 levels significantly increased (P= 0.04 and 0.03) after exercise. LPS induced IL-6 production was only marginally further increased by exercise. In conclusion, the ILCS system provided a reliable ex vivo method, showing common as well as subject specific features in the time course of the immune modulation caused by the LIST protocol. This system will be useful for studies of the elderly, where cytokine standardisation is notoriously difficult.

Plasma cytokine profiles in elderly humans.

It is known that as we age, immune dysregulation often occurs, leading to failing health, and increased susceptibility to a number of different diseases. In this study we have investigated plasma cytokine profiles in order to identify immune markers of ageing. Plasma samples were obtained from 138 participants of the Swedish longitudinal NONA study (aged 86, 90 and 94 years) and 18 healthy Swedish volunteers (aged between 32 and 59 years). Our results show significantly increased levels of the pro-inflammatory cytokine interleukin-6 (P<0.0001) and soluble intercellular adhesion molecule-1 (P<0.0001) in the elderly group. The anti-inflammatory cytokine interleukin-10 did not alter with age whereas active (naturally processed) transforming growth factor-beta levels were significantly (P<0.0001) increased in the elderly group. No difference was observed between males and females. These data suggest that there are measurable changes in cytokine profiles with ageing with increased levels of potentially harmful molecules, which may contribute to immune alterations and declining health in the elderly population.

Antioxidants may contribute in the fight against ageing: an in vitro model.

Elderly humans have altered cellular redox levels and dysregulated immune responses, both of which are key events underlying the progression of chronic degenerative diseases of ageing, such as atherosclerosis and Alzeimer's disease. Poorly maintained cellular redox levels lead to elevated activation of nuclear transcription factors such as NFkB and AP-1. These factors are co-ordinately responsible for a huge range of extracellular signalling molecules responsible for inflammation, tissue remodelling, oncogenesis and apoptosis, progessess that orchestrate many of the degenerative processess associated with ageing. It is now clear that levels of endogenous anti-oxidants such as GSH decrease with age. This study aimed to investigate the potential of exogenous anti-oxidants to influence inflammatory responses and the ageing process itself. We investigated the potential of the dietary antioxidant, quercetin, to reverse the age related influences of GSH depletion and oxidative stress using in vitro human umbilical vein endothelial cells (HUVEC) and human skin fibroblast (HSF) cell models. Oxidative stress-induced inflammatory responses were investigated in a GSH depletion and a Phorbol 12-myristate 13-acetate (PMA)-induced stress model. As measured with a sensitive HPLC fluorescence method, GSH in HUVEC was depleted by the addition of L-buthionine-[S,R]-sulfoxiniine (BSO), a gamma-glutamylcysteine synthetase inhibitor, to the culture medium at a concentration of 0.25 mM. Time course studies revealed that the GSH half-life was 4.6 h in HUVEC. GSH depletion by BSO for 24 h led to a slight increase in intracellular adhesion molecule - 1 (ICAM1) expression and prostaglandin E2 (PGE2) secretion in both types of cells. However, GSH depletion markedly enhanced PMA-induced ICAM and PGE2 production in HUVEC. Responses were progressively elevated following prolonged BSO treatment. Inhibition studies showed that 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a protein kinase C (PKC) inhibitor, not only abolished most of PMA-induced ICAM-1 expression and PGE2, production, but also eliminated GSH depletion-enhanced PMA stimulation. This enhancement was also inhibited by supplementation with quercetin. The results clearly demonstrate that GSH depletion increased the susceptibility of vascular endothelial cells and fibroblasts to oxidative stress associated inflammatory stimuli. This increased in vitro susceptibility may be extrapolated to the in vivo situation of ageing, providing a useful model to study the influence of micronutrients on the ageing process. In conclusion, these data suggest that dietary antioxidants could play a significant role in the reduction of inflammatory responses.

A proinflammatory role for IL-18 in rheumatoid arthritis.

IL-18 is a novel cytokine with pleiotropic activities critical to the development of T-helper 1 (Th1) responses. We detected IL-18 mRNA and protein within rheumatoid arthritis (RA) synovial tissues in significantly higher levels than in osteoarthritis controls. Similarly, IL-18 receptor expression was detected on synovial lymphocytes and macrophages. Together with IL-12 or IL-15, IL-18 induced significant IFN-gamma production by synovial tissues in vitro. IL-18 independently promoted GM-CSF and nitric oxide production, and it induced significant TNF-alpha synthesis by CD14(+) macrophages in synovial cultures; the latter effect was potentiated by IL-12 or IL-15. TNF-alpha and IFN-gamma synthesis was suppressed by IL-10 and TGF-beta. IL-18 production in primary synovial cultures and purified synovial fibroblasts was, in turn, upregulated by TNF-alpha and IL-1beta, suggesting that monokine expression can feed back to promote Th1 cell development in synovial membrane. Finally, IL-18 administration to collagen/incomplete Freund's adjuvant-immunized DBA/1 mice facilitated the development of an erosive, inflammatory arthritis, suggesting that IL-18 can be proinflammatory in vivo. Together, these data indicate that synergistic combinations of IL-18, IL-12, and IL-15 may be of importance in sustaining both Th1 responses and monokine production in RA.

Epidermal Langerhans cell apoptosis is induced in vivo by nonanoic acid but not by sodium lauryl sulphate.

Exposure to irritants may cause chronic irritant contact dermatitis (ICD), characterized by irregular epidermal thickening and a predominantly dermal mononuclear cell infiltrate. The mechanisms involved, and why only certain individuals are affected, are not clearly understood. Different irritants may trigger different cellular and molecular interactions between resident skin cells and recruited inflammatory cells. In some individuals these interactions may become self-perpetuating resulting in persistent inflammation in the absence of continued exposure. This study examined Langerhans cell (LC) density in clinically normal skin of 46 patients with chronic ICD and 10 healthy individuals, and compared the action of the two irritants nonanoic acid (NA) and sodium lauryl sulphate (SLS) on the LCs and keratinocytes of clinically normal skin in patients with chronic ICD. There was a higher number of LCs/mm basement membrane in patients compared with controls, although there was no difference in the number of dendrites/LC nor in dendrite length. SLS induced keratinocyte proliferation after 48 h exposure, had no effect on LC number or distribution, and induced keratinocyte apoptosis after 24 and 48 h exposure. In contrast, NA decreased keratinocyte proliferation after 24 h exposure but this returned to basal levels after 48 h, and induced epidermal cell apoptosis after only 6 h exposure. NA dramatically decreased LC number after 24 and 48 h exposure, which was accompanied by basal redistribution and decreased dendrite length. Most significantly, NA induced apoptosis in over half of the LCs present after 24 and 48 h exposure.

Epidermal keratinocyte production of interferon-gamma immunoreactive protein and mRNA is an early event in allergic contact dermatitis.

Previous work has indicated the importance of cytokine cascades in the induction of contact dermatitis, but there is little information on the cellular localization of cytokines in human skin, particularly during the early phases of the inflammatory response to contact allergens. Using in situ hybridization for mRNA and immunocytochemistry on biopsies from a series of 16 patients with known allergic contact dermatitis, we examined the kinetics of early cytokine production after challenge with relevant or irrelevant antigen. We show that epidermal keratinocytes from patients challenged in vivo with allergen, but not irrelevant antigen, rapidly synthesize (within 4 h) mRNA for interferon-gamma and produce immunoreactive interferon-gamma. Interleukin-1alpha and interleukin-8 mRNA were also detected but showed no correlation with relevant antigen challenge. This study demonstrates that keratinocytes can produce interferon-gamma and that this production is linked to challenge with relevant antigen in allergic contact dermatitis. These findings indicate that keratinocytes may amplify allergen-specific T-lymphocyte-triggered interferon-gamma dependent responses and might partially explain the speed of reaction in this common disease and other delayed hypersensitivity reactions involving the skin.