Robert N. Barker

Antenatal determinants of neonatal immune responses to allergens

Background The environmental factors responsible for recent increases in the prevalence of asthma and atopic disease have been assumed to act after birth. Their possible effects on fetal immune development in utero have not been investigated systematically, although sensitization to allergens may occur before birth.

Suppressor of cytokine signaling (SOCS)1 is a key determinant of differential macrophage activation and function

Macrophages become activated by their environment and develop polarized functions: classically activated (M1) macrophages eliminate pathogens but can cause tissue injury, whereas alternatively activated (M2) macrophages promote healing and repair. Mechanisms directing polarized activation, especially in vivo, are not understood completely, and here, we examined the role of SOCS proteins. M2 macrophages activated in vitro or elicited by implanting mice i.p. with the parasitic nematode Brugia malayi display a selective and IL‐4‐dependent up‐regulation of SOCS1 but not SOCS3. Using siRNA‐targeted knockdown in BMDM, we reveal that the enhanced SOCS1 is crucial for IL‐4‐induced M2 characteristics, including a high arginase I:iNOS activity ratio, suppression of T cell proliferation, attenuated responses to IFN‐γ/LPS, and curtailed SOCS3 expression. Importantly, SOCS1 was essential in sustaining the enhanced PI3K activity that drives M2 activation, defining a new regulatory mechanism by which SOCS1 controls M2 polarization. By contrast, for M1 macrophages, SOCS1 was not only an important regulator of proinflammatory mediators (IL‐6, IL‐12, MHC class II, NO), but critically, for M1, we show that SOCS1 also restricted IL‐10 secretion and arginase I activity, which otherwise would limit the efficiency of M1 macrophage proinflammatory responses. Together, our results uncover SOCS1, not only as a feedback inhibitor of inflammation but also as a critical molecular switch that tunes key signaling pathways to effectively program different sides of the macrophage balance.

Immunologically ignorant autoreactive T cells, epitope spreading and repertoire limitation

The factors that may cause antigen-presenting cells to alter the pattern of protein processing and presentation to autoreactive T cells, and thereby stimulate autoimmune disease, are currently under debate. In this article, Chris Elson and colleagues suggest that cytokines associated with T helper 1 (Th1) cells alter the processing of proteins and that this effect can be counteracted by Th2-associated cytokines.

Regulatory T Cells Secreting IL-10 Dominate the Immune Response to EBV Latent Membrane Protein 1

Viruses exploit a number of strategies to evade immune recognition. In this study, we describe a novel mechanism by which EBV, rather than avoiding detection, subverts the immune response by stimulating regulatory T cells that secrete IL-10. Human PBMC from all EBV-seropositive, but not -seronegative, donors responded to both purified latent membrane protein 1 and the corresponding immunodominant peptides with high levels of IL-10 secretion by CD4+ T cells. These IL-10 responses, characteristic of T regulatory 1 cells, inhibited T cell proliferation and IFN-γ secretion induced by both mitogen and recall Ag. It was confirmed that the inhibition was IL-10 dependent by the use of neutralizing Ab. The deviation of the immune response toward suppression is likely to be important in maintaining latency and EBV-associated tumors.

The Fine Specificity and Cytokine Profile of T-Helper Cells Responsive to the α3 Chain of Type IV Collagen in Goodpasture’s Disease

Goodpastures disease is a severe nephritis characterized by autoantibodies to the alpha3 chain of type IV collagen, alpha3(IV)NC1, in the glomerular basement membrane. The disease is very strongly associated with HLA-DR15, the affinities of alpha3(IV)NC1 peptides for DR15 are known, and elution experiments have identified major naturally processed sequences. Here, the fine specificity and cytokine profile of alpha3(IV)NC1-reactive T cells from patients with Goodpastures disease is defined. Peripheral blood mononuclear cells from patients at diagnosis proliferated in response to significantly more peptides (chi(2) = 8.6, P = 0.004) from a panel spanning the sequence of alpha3(IV)NC1 than did those from control DR15-positive donors and were highly focused (P = 0.0002, binomial distribution) on two peptides, alpha3(71-90) and alpha3(131-150). Some peptides induced interferon-gamma, but none induced IL-4. Resolution of disease was accompanied by a striking deviation of the responses from proliferation to secretion of the T-regulatory cytokine IL-10, and addition of neutralizing antibody confirmed that such IL-10 production was suppressive. The affinity of the peptides for DR15 molecules was positively correlated (chi(2) = 14.6, P = 0.00067) with the ability to elicit proliferation. However, unlike foreign antigens, this hierarchy is not due to responses against the major naturally processed peptides, which rarely stimulated proliferation and which have only intermediate affinity for DR15 molecules. It is inferred that the helper response to alpha3(IV)NC1 in Goodpastures disease is dominated by epitopes that are normally inefficiently presented because of processing constraints.

Antigen presentation by macrophages is enhanced by the uptake of necrotic, but not apoptotic, cells

The aim of this study was to determine whether phagocytosis of necrotic or apoptotic cells affects antigen presentation by murine bone marrow‐derived macrophages. After uptake of necrotic neutrophils, macrophages were able to stimulate significantly higher T cell proliferation in vitro against both the recall antigen albumin and the mitogen concanavalin A. No such effect was seen following phagocytosis of apoptotic neutrophils. Flow cytometry revealed that, within 4h of ingestion, macrophages that had taken up the necrotic cells expressed higher levels of CD40 than those that had phagocytosed apoptotic cells. Macrophage cultures pulsed with apoptotic, but not necrotic, neutrophils contained higher levels of transforming growth factor β1, but lower concentrations of tumour necrosis factor α, compared to untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic neutrophils co‐stimulate T cells with greater efficiency due to rapid CD40 up‐regulation, whereas those that have ingested apoptotic cells are not only ineffective in co‐stimulation, but also secrete inhibitory cytokine.

Differential Effects of Necrotic or Apoptotic Cell Uptake on Antigen Presentation by Macrophages

The induction of pathogenic immune responses may be dependent on the immune system receiving ‘danger’ signals resulting from tissue damage, rather than tolerogenic stimuli associated with normal cell turnover. The aim was to test this hypothesis by comparing the effects of the uptake of necrotic and apoptotic cells on the ability of antigen-presenting cells (APC) to stimulate immune responses in vitro. The experiments focused on presentation by the macrophage, which is the main cell type adapted for clearing cellular debris in vivo. Murine bone marrow-derived macrophages were pulsed with neutrophils that had been rendered apoptotic or necrotic, and tested for the ability to induce T cell responses. The macrophages that had taken up necrotic, but not apoptotic, cells were able to stimulate recall proliferation by ovalbumin-specific T cells. Furthermore, the response to the mitogen concanavalin A (Con A) was more than 6 times higher when macrophages had been pulsed with necrotic, in comparison with apoptotic, cells. In control experiments, macrophages that had not been exposed to dying neutrophils stimulated weak responses to ovalbumin and Con A. To determine why the uptake of apoptotic and necrotic cells exert opposing effects on the ability of macrophages to stimulate T cells, the expression of costimulatory molecules by treated macrophages, and their production of potentially immunomodulatory cytokines were measured. Flow cytometry revealed that macrophages that had taken up necrotic, but not apoptotic, neutrophils expressed increased levels of CD40 compared to untreated controls within 4 h. Macrophages pulsed with apoptotic cells secreted higher levels of transforming growth factor-β1 than those ingesting necrotic cells or untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic cell debris present antigens to T lymphocytes with greater efficiency due to transient CD40 upregulation, whereas those that have ingested apoptotic cells are ineffective APC since they secrete inhibitory cytokine.

In utero priming of allergen‐specific helper T cells

Background Allergic diseases are major health problems in developed countries. Cord blood mononuclear cells (CBMC) at birth can proliferate after stimulation with allergen and this has led to the widespread view that the sensitization of the fetal immune system by allergens is a key determinant in establishing immunological bias towards allergy. However, the notion that the immune system can be primed by allergen in utero remains unproven. Determination of the CD45 isoform of responding T helper cells is an established method of determining the activation status of responding T helper cells because unsensitized cells express CD45RAhigh and previously sensitized cells CD45ROhigh.

Macrophages: Promising Targets for the Treatment of Atherosclerosis

Atherosclerosis is now recognised as a chronic inflammatory disease occurring within the artery wall and ultimately responsible for myocardial infarction, stroke and peripheral vascular disease. A crucial step in atherogenesis is the infiltration of monocytes into the subendothelial space of large arteries where they differentiate into macrophages and become functionally active. Macrophage accumulation within plaques is a hallmark of all stages of atherosclerosis, indeed recent studies have shown their presence has the potential to act as a non-invasive marker of disease activity and plaque stability. Activated macrophages are major players in all stages of lesion development. They not only accumulate lipids but also express effector molecules that are pro-inflammatory, cytotoxic and chemotactic. Furthermore, they secrete enzymes that degrade extracellular matrix leading to plaque destabilisation and increased risk of rupture. However, macrophages are heterogeneous and when appropriately activated they have the potential to drive tissue remodelling and ultimately vascular repair. Pharmacological modulation of macrophage activities therefore represents an important strategy for the prevention and treatment of atherosclerosis and other inflammatory diseases. The aim of this review is to give a brief overview of our current understanding of macrophage activation, distribution and function within inflamed tissue. This will provide the basis for highlighting already available and future methods to exploit specifically activated macrophages as diagnostic and therapeutic targets for atherosclerosis.

A critical role for suppressor of cytokine signalling 3 in promoting M1 macrophage activation and function in vitro and in vivo.

Macrophages respond to their microenvironment and develop polarized functions critical for orchestrating appropriate inflammatory responses. Classical (M1) activation eliminates pathogens while alternative (M2) activation promotes regulation and repair. M1 macrophage activation is strongly associated with suppressor of cytokine signalling 3 (SOCS3) expression in vitro, but the functional consequences of this are unclear and the role of SOCS3 in M1‐macrophage polarization in vivo remains controversial. To address these questions, we defined the characteristics and function of SOCS3‐expressing macrophages in vivo and identified potential mechanisms of SOCS3 action. Macrophages infiltrating inflamed glomeruli in a model of acute nephritis show significant up‐regulation of SOCS3 that co‐localizes with the M1‐activation marker, inducible nitric oxide synthase. Numbers of SOCS3hi‐expressing, but not SOCS1hi‐expressing, macrophages correlate strongly with the severity of renal injury, supporting their inflammatory role in vivo. Adoptive transfer of SOCS3‐short interfering RNA‐silenced macrophages into a peritonitis model demonstrated the importance of SOCS3 in driving production of pro‐inflammatory IL‐6 and nitric oxide, while curtailing expression of anti‐inflammatory IL‐10 and SOCS1. SOCS3‐induced pro‐inflammatory effects were due, at least in part, to its role in controlling activation and nuclear accumulation of nuclear factor‐κB and activity of phosphatidylinositol 3‐kinase. We show for the first time that SOCS3 also directs the functions of human monocyte‐derived macrophages, including efficient M1‐induced cytokine production (IL‐1β, IL‐6, IL‐23, IL‐12), attenuated signal transducer and activator of transcription 3 activity and ability of antigen‐loaded macrophages to drive T‐cell responses. Hence, M1‐associated SOCS3 was a positive regulator of pro‐inflammatory responses in our rodent models and up‐regulated SOCS3 is essential for effective M1‐macrophage activation and function in human macrophages.