Aging impairs peritoneal but not bone marrow-derived macrophage phagocytosis

Aging results in deterioration of the immune system, which is associated with increased susceptibility to infection and impaired wound healing in the elderly. Phagocytosis is an essential process in both wound healing and immune defence. As such, age-related impairments in phagocytosis impact on the health of the elderly population. Phagocytic efficiency in peritoneal macrophages, bone marrow-derived macrophages and bone marrow monocytes from young and old mice was investigated. Aging significantly impaired phagocytosis by peritoneal macrophages, both in vitro and in vivo. However, bone marrow-derived macrophages and bone marrow monocytes did not exhibit age-related impairments in phagocytosis, suggesting no intrinsic defect in these cells. We sought to investigate underlying mechanisms in age-related impairments in phagocytosis by peritoneal macrophages. We hypothesized that microenvironmental factors in the peritoneum of old mice impaired macrophage phagocytosis. Indeed, macrophages from young mice injected into the peritoneum of old mice exhibited impaired phagocytosis. Proportions of peritoneal immune cells were characterized, and striking increases in numbers of T cells, B1 and B2 cells were observed in the peritoneum of old mice compared with young mice. In addition, B cell-derived IL-10 was increased in resting and LPS-activated peritoneal cell cultures from old mice. These data demonstrate that aging impairs phagocytosis by tissue-resident peritoneal macrophages, but not by bone marrow-derived macrophages/monocytes, and suggest that age-related defects in macrophage phagocytosis may be due to extrinsic factors in the tissue microenvironment. As such, defects may be reversible and macrophages could be targeted therapeutically in order to boost immune function in the elderly.

Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation.

METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1β response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover.

RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1β release from macrophages, through lysosomal destabilization. IL-1β secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1β release was significantly reduced in primary macrophages from ctss(-/-) mice.

CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1β release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

Secreted Proteins from the Helminth Fasciola hepatica Inhibit the Initiation of Autoreactive T Cell Responses and Prevent Diabetes in the NOD Mouse

Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-γ secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFβ and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.

A phase Ib trial of Docetaxel, Carboplatin and Erlotinib in ovarian, fallopian tube and primary peritoneal cancers.

The safety and maximum tolerated dose (MTD) of erlotinib with docetaxel/carboplatin were assessed in patients with ovarian cancer. Chemonaive patients received intravenous docetaxel (75 mg m(-2)) and carboplatin (area under the curve 5) on day 1 of a 3-week cycle, and oral erlotinib at 50 (cohort 1), 100 (cohort 2a) or 75 mg day(-1) (cohort 2b) for up to six cycles. Dose-limiting toxicities were determined in cycle 1. Forty-five patients (median age 59 years) received treatment. Dose-limiting toxicities occurred in 1/5/5 patients (cohorts 1/2a/2b). The MTD of erlotinib in this regimen was determined to be 75 mg day(-1) (cohort 2b; the erlotinib dose was escalated to 100 mg day(-1) in 11 out of 19 patients from cycle 2 onwards). Neutropaenia was the predominant grade 3/4 haematological toxicity (85/100/95% respectively). Common non-haematological toxicities were diarrhoea, fatigue, nausea and rash. There were five complete and seven partial responses in 23 evaluable patients (52% response rate). Docetaxel/carboplatin had no measurable effect on erlotinib pharmacokinetics. In subsequent single-agent maintenance, erlotinib was given at 100-150 mg day(-1), with manageable toxicity, until tumour progression. Further investigation of erlotinib in epithelial ovarian carcinoma may be warranted, particularly as maintenance therapy

Liver fluke (Fasciola hepatica)-derived peptides activate rat peritoneal mast cells

Short peptides with sequences derived from those found in the tegumental antigen of Fasciola hepatica have been synthesised. Incubation of some of these peptides with rat peritoneal mast cells resulted in the degranulation of the cells as measured by a histamine release assay. This activity was shown to be associated with the proline-lysine-proline motif, which is responsible for the induction of mast cell degranulation by the mammalian bioactive peptide substance P. Studies on the mode of action of the fluke-derived peptide indicated that it was operating through the same biochemical pathways as substance P. The implications of these findings for the development of immune responses during parasite infections are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

The response of macrophages to particles of resorbable polymers and their degradation products

Alpha polyesters such as poly(L-lactide) and poly(glycolide) are biodegradable materials used in fracture fixation and they need to be assessed for problems associated with their degradation products. This study has compared cell responses to low molecular weight poly(L-lactide) particles, lactate monomer, poly(glycolide) particles and glycolic acid at cytotoxic and sub-cytotoxic concentrations. Murine macrophages were cultured in vitro and the release of lactate dehydrogenase (LDH), prostaglandin E-2 (PGE(2)) and interleukin-1 alpha IL-1alpha was measured following the addition of particles or monomer. Experiments revealed that both the poly(L-lactide) and poly(glycolide) particles gave rise to dose dependent increases in LDH release and an increase in IL-1alpha and PGE(2) release. Comparisons of the poly(L-lactide) particles to the poly(glycolide) particles did not reveal any differences in their stimulation of LDH, IL-1alpha and PGE(2) release. The lactate and glycolate monomers did not increase PGE(2) or IL-1alpha release above control levels. There was no difference in biocompatibility between the poly(L-lactide) and poly(glycolide) degradation products both in particulate and monomeric form. (C) 2003 Kluwer Academic Publishers.