Robin Callard

CD40 Is Constitutively Expressed on Platelets and Provides a Novel Mechanism for Platelet Activation

Abstract— CD40 is a 48-kDa phosphorylated transmembrane glycoprotein belonging to the TNF receptor superfamily. CD40 has been demonstrated on a range of cell types, and it has an important role in adaptive immunity and inflammation. CD40 has recently been described on platelets but platelet activation by CD40 has not been described. In the present study, we use flow cytometry and immunoblotting to confirm that platelets constitutively express surface CD40. CD40 mRNA was undetectable, suggesting that the protein is synthesized early in platelet differentiation by megakaryocytes. Ligation of platelet CD40 with recombinant soluble CD40L trimer (sCD40LT) caused increased platelet CD62P expression, &agr;-granule and dense granule release, and the classical morphological changes associated with platelet activation. CD40 ligation also caused &bgr;3 integrin activation, although this was not accompanied by platelet aggregation. These actions were abrogated by the CD40L blocking antibody TRAP-1 and the CD40 blocking antibodies M2 and M3, showing that activation was mediated by CD40L binding to platelet CD40. &bgr;3 integrin blockade with eptifibatide had no effect, indicating that outside-in signaling via &agr;IIb&bgr;3 was not contributing to these CD40-mediated effects. CD40 ligation led to enhanced platelet-leukocyte adhesion, which is important in the recruitment of leukocytes to sites of thrombosis or inflammation. Our results support a role for CD40-mediated platelet activation in thrombosis, inflammation, and atherosclerosis.

Epicutaneous exposure to peanut protein prevents oral tolerance and enhances allergic sensitization.

Background Food allergies are an important cause of life‐threatening hypersensitivity reactions. Oral tolerance can be considered the default immune response to dietary antigens, with immune deviation resulting in allergic sensitization. However, primary sensitization to food allergens may not solely be through the gastrointestinal mucosa, as strong T‐helper type 2 (Th2)‐biased immunity can result from exposure to protein allergens on barrier‐disrupted skin.

Disruption of the stratum corneum allows potent epicutaneous immunization with protein antigens resulting in a dominant systemic Th2 response.

The skin is an important immunological organ with an outer protective layer, the stratum corneum forming a barrier between the skin‐associated lymphoid tissue and the environment. We show thatgently removing the stratum corneum with adhesive tape permits potent epicutaneous immunization to protein antigens. IL‐4 secretion by T cells from draining lymph nodes and high levels of specific IgE and IgG1 with no IgG2a showed that the immune responses induced following epicutaneous antigen exposure are strongly Th2 biased. Similar responses were obtained with different antigens and mousestrains. In contrast, subcutaneous immunization with antigen delivery into the dermis was less potent and gave predominantly Th1 responses. Removal of the stratum corneum increased expression of MHC class II, CD86, CD40, CD54 and CD11c on Langerhans cells, but did not cause them to migrate. Rapid migration from epidermis to draining lymph node was obtained, however, by exposure to antigen after removal of the stratum corneum, suggesting that maturation and migration of Langerhans cells are independently regulated events. These results suggest that antigen presentation by Langerhans cells gives predominantly Th2 responses. This may provide an explanation for allergic sensitization to some antigens. It may also be a useful non‐invasive, non‐adjuvant‐dependent method of vaccination.

Cytokines, Chaos, and Complexity

Mathematical models will not replace laboratory experimentation, but they do have an important complementary role for understanding the complexities of the cytokine network and other biological systems. Such models cannot incorporate every component of the network, as the model itself would then be impossible to analyze. Rather, they should be considered as virtual laboratories in which numerical experiments can be performed that could not otherwise be undertaken. Such virtual experiments can deal with more parameters than in vitro or in vivo experimentation and can give insights not available through more conventional investigations. A dialogue between the two disciplines is required. The biologists should define the problem and provide quantitative information about the system from which a model can be constructed. Mathematical analysis of the model can then make predictions about the behavior of the biological system that can be tested in the laboratory. The more refined data obtained from these experiments can then be fed back into the model. By this iterative process, mathematics and biology can combine to give a deeper understanding of the cytokine network and other biological processes.∣∣To whom correspondence should be addressed (e-mail: rcallard@ich.ucl.ac.uk).

Ranked prediction of p53 targets using hidden variable dynamic modeling

Full exploitation of microarray data requires hidden information that cannot be extracted using current analysis methodologies. We present a new approach, hidden variable dynamic modeling (HVDM), which derives the hidden profile of a transcription factor from time series microarray data, and generates a ranked list of predicted targets. We applied HVDM to the p53 network, validating predictions experimentally using small interfering RNA. HVDM can be applied in many systems biology contexts to predict regulation of gene activity quantitatively.

Early postoperative monocyte deactivation predicts systemic inflammation and prolonged stay in pediatric cardiac intensive care

ObjectiveSepsis and systemic inflammatory response syndrome (SIRS) are major causes of morbidity and mortality after cardiopulmonary bypass. Attempts to suppress proinflammatory mediators have failed to improve outcomes in sepsis or in patients undergoing cardiopulmonary bypass. Recent work in adult patients has suggested that the balance between pro- and anti-inflammatory mediators is more important than the level of proinflammatory response alone. This balance may be reflected by the expression of monocyte human lymphocyte antigen (HLA)-DR, with low concentrations indicating an excess of anti-inflammatory stimuli and relative immunodeficiency. We investigated the relationship between monocyte HLA-DR expression and the subsequent development of sepsis/SIRS in children undergoing cardiopulmonary bypass. DesignA prospective, observational, clinical study. SettingA tertiary pediatric cardiac center. PatientsEighty-two infants and children undergoing elective cardiac surgery between March and December 1999. Measurements and Main ResultsMonocyte HLA-DR expression was assessed before and after surgery and was found to be related to the length of hospital stay and the development of complications including sepsis/SIRS. The inflammatory insult of cardiopulmonary bypass decreased monocyte HLA-DR expression in all children. Lowest concentrations were seen within 72 hrs of surgery and were significantly lower in cases that subsequently required prolonged intensive care support (p < .0001, Mann-Whitney). HLA-DR expression on <60% of circulating monocytes was associated with a greatly increased risk of later (minimum 4 days) development of sepsis/SIRS (odds ratio, 12.9; 95% confidence interval, 3.4–47.5). Low HLA-DR was an independent predictor for the development of sepsis/SIRS after correction for age, bypass time, complexity of surgery, Paediatric Index of Mortality, and surgeon on multiple logistic regression analysis. ConclusionsPatients with decreased HLA-DR in the early postoperative period represent a subpopulation at greatly increased risk of later sepsis/SIRS. Such patients may benefit from strategies aimed to reduce this risk.

Biological function of CD40 on human endothelial cells: costimulation with CD40 ligand and interleukin‐4 selectively induces expression of vascular cell adhesion molecule‐1 and P‐selectin resulting in preferential adhesion of lymphocytes

The expression of adhesion molecules on vascular endothelial cells determines the pattern of migration and extravasation of leucocytes in inflammation and immunity. Here we show that costimulation with CD40 ligand (CD40L) and interleukin (IL)‐4 (or IL‐13) gives rise to a unique pattern of adhesion molecule expression by human umbilical vein endothelial cells (HUVEC). CD40 ligation alone enhanced expression of vascular cell adhesion molecule‐1 (VCAM‐1), intracellular adhesion molecule‐1 (ICAM‐1) and E‐selectin whereas IL‐4 and IL‐13 increased expression of VCAM‐1 and P‐selectin but not ICAM‐1 or E‐selectin. When IL‐4 and CD40L were combined there was an additional increase of both VCAM‐1 and P‐selectin, but ICAM‐1 and E‐selectin were both inhibited. The combined effects of IL‐4 and CD40L signalling were not the result of altered response kinetics, enhanced sensitivity of the endothelium, or increased expression of CD40 or the IL‐4 receptor. The rise in VCAM‐1 expression induced by combined IL‐4 and CD40L stimulation was slower and more sustained than with tumour necrosis factor‐α (TNF‐α) and occurred only on a subset (75–80%) of the endothelial cell population compared to 100% with TNF‐α. Costimulation with IL‐4 and CD40L increased adhesion of T cells and B cells above levels obtained with either signal alone, but decreased adhesion of neutrophils. Furthermore, CD40 and IL‐4 synergistically increased IL‐6 but decreased IL‐8 production by HUVEC. These results show that interactions between IL‐4 and CD40 on endothelial cells give rise to specific patterns of adhesion molecule expression and cytokine production that may have important implications for lymphocyte and neutrophil migration and function at sites of inflammation.

Quantifying the development of the peripheral naive CD4+ T-cell pool in humans

What are the rules that govern a naive T cells prospects for survival or division after export from the thymus into the periphery? To help address these questions, we combine data from existing studies with robust mathematical models to estimate the absolute contributions of thymopoiesis, peripheral division, and loss or differentiation to the human naive CD4+ T-cell pool between the ages of 0 and 20 years. Despite their decline in frequency in the blood, total body numbers of naive CD4+ T cells increase throughout childhood and early adulthood. Our analysis shows that postthymic proliferation contributes more than double the number of cells entering the pool each day from the thymus. This ratio is preserved with age; as the thymus involutes, the average time between naive T-cell divisions in the periphery lengthens. We also show that the expected residence time of naive T cells increases with time. The naive CD4+ T-cell population thus becomes progressively less dynamic with age. Together with other studies, our results suggest a complex picture of naive T-cell homeostasis in which population size, time since export from the thymus, or time since the last division can influence a cells prospects for survival or further divisions.

Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria.

ABSTRACT Interactions between dendritic cells (DCs) and microbial pathogens are fundamental to the generation of innate and adaptive immune responses. Upon stimulation with bacteria or bacterial components such as lipopolysaccharide (LPS), immature DCs undergo a maturation process that involves expression of costimulatory molecules, HLA molecules, and cytokines and chemokines, thus providing critical signals for lymphocyte development and differentiation. In this study, we investigated the response of in vitro-generated human DCs to a serogroup B strain of Neisseria meningitidis compared to an isogenic mutant lpxA strain totally deficient in LPS and purified LPS from the same strain. We show that the parent strain,lpxA mutant, and meningococcal LPS all induce DC maturation as measured by increased surface expression of costimulatory molecules and HLA class I and II molecules. Both the parent and lpxAstrains induced production of tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), and IL-6 in DCs, although the parent was the more potent stimulus. In contrast, high-level IL-12 production was only seen with the parent strain. Compared to intact bacteria, purified LPS was a very poor inducer of IL-1α, IL-6, and TNF-α production and induced no detectable IL-12. Addition of exogenous LPS to thelpxA strain only partially restored cytokine production and did not restore IL-12 production. These data show that non-LPS components of N. meningitidis induce DC maturation, but that LPS in the context of the intact bacterium is required for high-level cytokine production, especially that of IL-12. These findings may be useful in assessing components of N. meningitidis as potential vaccine candidates.

Neisseria meningitidis expressing lgtB lipopolysaccharide targets DC-SIGN and modulates dendritic cell function

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto‐N‐neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties  towards  DC.  These  properties  were  mediated by interaction of the GlcNAc(β1‐3)‐Gal(β1‐4)‐Glc‐R oligosaccharide outer core of lgtB LPS with the dendritic‐cell‐specific ICAM‐3 grabbing non‐integrin (DC‐SIGN) lectin receptor. Activation of DC‐SIGN with this novel oligosaccharide ligand skewed T‐cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.