Denis J. Reen

Lipopolysaccharide Induces Rapid Production of IL-10 by Monocytes in the Presence of Apoptotic Neutrophils

There is growing evidence that apoptotic neutrophils have an active role to play in the regulation and resolution of inflammation following phagocytosis by macrophages and dendritic cells. However, their influence on activated blood monocytes, freshly recruited to sites of inflammation, has not been defined. In this work, we examined the effect of apoptotic neutrophils on cytokine production by LPS-activated monocytes. Monocytes stimulated with LPS in the presence of apoptotic neutrophils for 18 h elicited an immunosuppressive cytokine response, with enhanced IL-10 and TGF-β production and only minimal TNF-α and IL-1β cytokine production. Time-kinetic studies demonstrated that IL-10 production was markedly accelerated in the presence of apoptotic neutrophils, whereas there was a sustained reduction in the production of TNF-α and IL-1β. This suppression of proinflammatory production was not reversible by depletion of IL-10 or TGF-β or by addition of exogenous IFN-γ. It was demonstrated, using Transwell experiments, that monocyte-apoptotic cell contact was required for induction of the immunosuppressive monocyte response. The response of monocytes contrasted with that of human monocyte-derived macrophages in which there was a reduction in IL-10 production. We conclude from these data that interaction between activated monocytes and apoptotic neutrophils creates a unique response, which changes an activated monocyte from being a promoter of the inflammatory cascade into a cell primed to deactivate itself and other cells.

Neonatal immunity: how well has it grown up?

Abstract Recent experiments have led to renewed interest in differences between immune responses in adults and neonates. Here, Stuart Marshall-Clarke and colleagues discuss and propose a model suggesting that differences at the B-cell level might play an important role in the impoverished responses and reported Th2 bias of neonates.

Human Recent Thymic Emigrants–Identification, Expansion, And Survival Characteristics

This study shows that, in humans at birth, circulating T cells represent recent thymic emigrants (RTEs) as reflected in their high level of expression of TCR excision circles. RTEs express “thymocyte-like” characteristics with regard to rapid rate of apoptosis. In the presence of common γ-chain cytokines, in particular IL-7, they show enhanced potential to survive, entry into cell cycle, and proliferation. Although common γ-chain cytokines were also potent antiapoptotic stimuli for mature adult-derived naive CD4+CD45RA+ T cells, these cells were refractory to IL-7-induced expansion in vitro. RTEs cultured with IL-7 could not reinduce recombination-activating gene-2 gene expression in vitro. These data suggest that postthymic naive T cells in the periphery during early life are at a unique stage in ontogeny as RTEs, during which they can undergo homeostatic regulation including expansion and survival in an Ag-independent manner while maintaining their preselected TCR repertoire.

IL‐7 promotes the survival and maturation but not differentiation of human post‐thymic CD4+ T cells

This study examines the influence of IL‐7 on post‐thymic CD4+ T cells using cord blood as a model system. Survival of naive cord blood T cells in the presence of IL‐7 alone was significantly prolonged by up‐regulating bcl‐2, thereby preventing apoptosis while maintaining maximal cell viability. Cultures without IL‐7 showed high rates of apoptosis resulting in 50 % cell death by day 5 of culture. Upon phorbol 12‐myristate 13‐acetate + ionomycin stimulation, accumulation of cytoplasmic IL‐2 was similar to that observed in freshly isolated cells, but no IL‐4‐ or IFN‐γ‐positive cells were detected. IL‐7 maintained the naive T cells in a quiescent state expressing the CD45RA antigen. A significant finding was the loss of CD38 antigen expression on the naive cord blood T cells to levels similar to that observed on adult naive T cells. In contrast to the reduced proliferative response of fresh cord blood T cells to anti‐CD2 + CD28 stimulation, the proliferative response of IL‐7‐treated cells was similar to that of adult naive T cells. This study shows that as well as maintaining the naive T cell pool by enhancing cell survival and up‐regulating bcl‐2 expression, IL‐7 also functions as a maturation factor for post‐thymic naive T cells.

Interleukin 10, produced in abundance by human newborn T cells, may be the regulator of increased tolerance associated with cord blood stem cell transplantation

Summary. The use of human umbilical cord blood as an alternative source of stem cells to bone marrow for the reconstitution of the immune system is associated with less frequent and less severe incidence of graft‐versus‐host disease (GVHD). This study focuses on aspects of cord blood T‐cell biology that may contribute to a perceived increased tolerance associated with the neonatal immune response. A skewing of the T‐helper (Th)1/Th2 phenotype of cord blood T cells towards a Th2 response has frequently been cited as a possible cause. In this study, primary and repeated stimulation via the T‐cell receptor (TCR) complex induced a Th0‐type cytokine response, with both adult and cord blood‐derived naïve T cells producing interferon γ (IFN‐γ), interleukin 4 (IL‐4) and IL‐5. IL‐10 was induced in cord blood T‐cell cultures during primary stimulation, while adult T cells began to secrete IL‐10 only after repeated stimulation. The presence of the antigen‐presenting cell (APC)‐derived cytokine IL‐1β inhibited IL‐10 production by cord blood cells. The effects of IL‐12 and IL‐4 on T‐cell cytokine responses were also examined. In addition to their differential Th1/Th2 skewing effects on cord and adult T cells, both cytokines augmented IL‐10 production in both T‐cell populations. These findings demonstrate that cord blood T cells may secrete large amounts of the anti‐inflammatory cytokine IL‐10 and that the presence of IL‐1β or Th1/Th2 skewing cytokines can regulate its production. This data provides support for the recognized tolerant nature of the newborn immune response that may contribute to the reduced incidence of GVHD associated with cord blood transplantation.

Abnormal innervation and altered nerve growth factor messenger ribonucleic acid expression in ureteropelvic junction obstruction.

The pathophysiology of ureteropelvic junction obstruction is unknown. Using specific antibodies, we studied specimens from 35 cases of ureteropelvic junction obstruction and 32 of normal ureteropelvic junction by immunohistochemistry using protein gene product 9.5 (a general neuronal marker), S100 (a supporting cell marker), synaptophysin (a neuromuscular junction marker) and nerve growth factor receptor. Nerve growth factor expression was examined at the messenger ribonucleic acid (mRNA) level using reverse transcription-polymerase chain reaction technique in 11 ureteropelvic junction obstruction specimens and 7 controls. The most striking finding was the marked reduction of protein gene product 9.5, synaptophysin and nerve growth factor receptor staining positive nerve fibers in the muscle layers of ureteropelvic junction obstruction compared to the normal ureteropelvic junction. Supporting nerve cell fibers (S100) were preserved in cases of ureteropelvic junction obstruction and normal ureteropelvic junction. A significantly less intense signal for nerve growth factor mRNA was found in the ureteropelvic junction obstruction specimens compared to normal ureteropelvic junction. These findings suggest that defective innervation may have an important role in the pathogenesis of ureteropelvic junction obstruction, and decreased nerve growth factor mRNA expression may be important in the etiology of ureteropelvic junction obstruction.

Activated Human Neonatal CD8+ T Cells Are Subject to Immunomodulation by Direct TLR2 or TLR5 Stimulation

In conditions of optimal priming, the neonate possess competency to mount quantitatively adult-like responses. Vaccine formulations containing sufficiently potent adjuvants may overcome the neonates’ natural tendency for immunosuppression and provoke a similarly robust immune response. TLR expression on T cells represents the possibility of directly enhancing T cell immunity. We examined the ex vivo responsiveness of highly purified human cord blood-derived CD8+ T cells to direct TLR ligation by a repertoire of TLR agonists. In concert with TCR stimulation, only Pam3Cys (palmitoyl-3-Cys-Ser-(Lys)4) and flagellin monomers significantly enhanced proliferation, CD25+ expression, IL-2, IFN-γ, TNF-α, and intracellular granzyme B expression. TLR2 and TLR5 mRNA was detected in the CD8+ T cells. Blocking studies confirmed that the increase in IFN-γ production was by the direct triggering of surface TLR2 or TLR5. The simultaneous exposure of CD8+ T cells to both TLR agonists had an additive effect on IFN-γ production. These data suggest that a combination of the two TLR ligands would be a potent T cell adjuvant. This may represent a new approach to TLR agonist-based adjuvant design for future human neonatal vaccination strategies requiring a CD8+ component.

Modulation of immune cell function by polyunsaturated fatty acids.

The n–3 and n–6 polyunsaturated fatty acids (PUFAs) are essential dietary constituents. They are important as a source of energy, as structural components of cell membranes, and as signalling molecules. They have been demonstrated to be potent modulators of the immune response, and research has endeavoured to optimise the ratio of n–3 to n–6 PUFAs in the lipid component of total parenteral nutrition (TPN) to optimise their beneficial effects in the clinical setting. Critically ill neonates on TPN have an increased incidence of sepsis, and additional studies have determined that lipid emulsions depress various elements of cellular immune responses in monocytes, lymphocytes, and neutrophils. It has been proposed that PUFAs may mediate their manifold effects through the modification of eicosanoid production and by directly or indirectly modifying intracellular signal transduction pathways, including the alteration of gene transcription, in various tissues. They are susceptible to lipid peroxidation, and there is evidence that the products of this process may result in cell death by apoptosis, a nonphlogistic homeostatic process of cell deletion. PUFAs have been shown to induce apoptosis in primary lymphocytes, colonic mucosal cells, and various cell lines. Additionally, our laboratory has shown them to be potent inducers of apoptosis in neonatal monocytes. This may represent a novel mechanism whereby PUFAs may modify the immune response.

Early induction of IL-6 in infants undergoing major abdominal surgery

Cytokines are immunoregulatory molecules that are important mediators of the host response to stress and infection. Infants and children undergoing major surgery are particularly at risk of developing sepsis and have altered metabolic responses to surgical stress compared to adults. We have investigated the temporal sequence of cytokine responses in six infants (mean age, 11 +/- 7.5 months) undergoing pull-through operation for Hirschsprungs disease and correlated them with hemodynamic and biochemical parameters. Tumor necrosis factor (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) were measured by ELISA preoperatively, intraoperatively (hourly), and 24 and 48 hours postoperatively. IL-6 levels increased significantly in all cases within 2 hours of commencement of the operation (P less than .01) and were maximal 24 hours postoperatively. No significant changes in IL-1 beta levels (mean range, 70 to 110 pg/mL) were seen in these patients. TNF levels were undetectable (less than 20 pg/mL) throughout the study. Cortisol levels were increased in all patients during operation. Serum C-reactive protein levels were first detected 24 hours postoperatively and continued to increase 48 hours postoperatively. Hemodynamically, heart rate increased during the first 3 hours of operation and correlated with increase in IL-6 levels. Blood pressure and temperature changes did not correlate with cytokine levels. This study identifies IL-6 as the earliest detectable cytokine response associated with major surgery in infants. It also suggests that IL-6 can be unregulated, independently of other cytokines, in response to surgical stress.

Inflammatory response in enterocolitis in the piebald lethal mouse model of Hirschsprung's disease.

ABSTRACT: Mucosal immune defense responses in two clinical forms of enterocolitis complicating congenital me-gacolon in the piebald lethal mouse model of Hirschsprungs disease were investigated. Significant histological and immunocytochemical differences were seen in the ganglionic segment of colon between piebald mice with early clinical onset of acute illness and piebald mice with late onset enterocolitis. The number of IgA-containing plasma cells infiltrating the lamina propria of the gut in the former group were significantly increased (p < 0.001) compared to the latter group of mice and healthy controls. This study suggests that the two forms of enterocolitis complicating congenital megacolon in piebald mice have different etiologies.