Neonatal CD8+ T-cell differentiation is dependent on interleukin-12.

Neonatal CD8(+) T-cell activation is significantly impaired compared with that in adults. Recent studies have demonstrated that interleukin (IL)-12 is necessary as a third signal, in addition to antigen and co-stimulation, to authorize the differentiation of naive CD8(+) T cells. We examined whether human neonatal CD8(+) T cells, which possess an exclusively naive T-cell phenotype, required a third signal to authorize a productive T-cell response. IL-12 enhanced activated naive CD8(+) T-cell survival, expansion, CD25 expression, and IL-2 production. Activated CD8(+) T cells produced interferon-γ and intracellular granzyme B and were cytotoxic only in the presence of IL-12. Sustained IL-12 signaling for 72 hours was required for optimal interferon-γ production. IL-12, in concert with T cell receptor (TCR) stimulation, sustained late-stage (48-72 hours) intracellular phosphorylation and particularly total protein levels of the proximal TCR components, Lck, and CD3ξ. The requirement for a third signal for productive human neonatal CD8(+) T-cell differentiation may have implications for neonatal vaccination strategies.

Activated human neonatal CD8+ T cells are subject to immunomodulation by direct TLR2 or TLR5 stimulation.

In conditions of optimal priming, the neonate possesses competency to mount quantitatively adult-like responses. Vaccine formulations containing sufficiently potent adjuvants may overcome the neonate's 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 Pam(3)Cys (palmitoyl-3-Cys-Ser-(Lys)(4)) and flagellin monomers significantly enhanced proliferation, CD25(+) expression, IL-2, IFN-gamma, TNF-alpha, and intracellular granzyme B expression. TLR2 and TLR5 mRNA was detected in the CD8(+) T cells. Blocking studies confirmed that the increase in IFN-gamma 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-gamma 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.

Neonatal human autologous dendritic cells pulsed with recombinant protein antigen prime the generation of non-polarized CD4 T-cell effectors.

The functional capability of human neonatal CD4 T cells to respond to vaccine antigens is frequently described as Th2 biased, but whether this is due to defective T-cell or antigen-presenting cell (APC) function is unclear. In this study, we used purified T cells and autologous monocyte-derived dendritic cells (MDDCs) as APCs to model primary and secondary neonatal CD4 T-cell responses in vitro to BBG2Na, a recombinant protein subunit vaccine candidate against respiratory syncytial virus (RSV). Neonatal MDDCs were phenotypically and functionally comparable to adult-derived MDDCs in terms of stimulatory capacity, longevity and ability to direct Th1 differentiation. When pulsed with BBG2Na, they induced antigen-specific neonatal CD4 T-cell proliferation. Analysis of cytokine production by quantitative real-time PCR showed significant production of IFN-gamma and IL-13 mRNA, analogous to the non-polarized primary cytokine mRNA response exhibited by both neonatal and adult naive CD4 T cells when primed by keyhole limpet haemocyanin. This contrasts with BBG2Na-activated adult CD45R0+ve memory CD4 T-cell responses, originally primed by natural RSV infection, which demonstrated a polarized Th1 cytokine profile. Importantly, on secondary stimulation, BBG2Na-primed neonatal CD4 T cells exhibited a 4-fold increase in antigen-specific proliferation and a 5-fold increase in IFN-gamma production. These data suggest that early life human CD4 T cells in vitro are intrinsically functionally capable of being primed by subunit vaccine candidate antigens such as BBG2Na, and differentiate into non-polarized rather than Th2 effectors.

Induction and modulation of apoptosis in neonatal monocytes by polyunsaturated fatty acids.

Polyunsaturated fatty acids (PUFAs), known modulators of the immune response, are the source of essential fatty acids in total parenteral nutrition-dependent patients. Critically ill infants on TPN have an increased incidence of sepsis, and lipid emulsions depress various immune functions. Recent studies have demonstrated that PUFAs induce apoptosis in various tissue cells in vitro and ex vivo. The susceptibility of neonatal monocytes, as major early effector cells in the host response to sepsis, to PUFA-mediated apoptosis and the mechanisms associated with PUFA-induced apoptosis were investigated. Both n-3 and n-6 PUFAs induced rapid, dose-dependent cell death in purified monocytes. Polyunsaturated fatty acids induced significant activation of upstream caspases 8 and 9 as well as caspase 3. The PUFA treatment resulted in a 4-fold increase in oxidative stress and a loss of monocyte mitochondrial potential compared with carrier controls (P < .05). The addition of cyclosporin, which blocks the development of mitochondrial transition pores, completely abolished the proapoptotic effects of PUFAs. Although Trolox (Sigma Aldrich) reduced PUFA-induced intracellular oxidative stress in neonatal monocytes, apoptosis was not blocked by this potent antioxidant. The data identify PUFAs as potent inducers of monocyte apoptosis, which can occur independently of the induction of oxidative stress, by using a mitochondrial dependent pathway. The TPN-dependent infant may be particularly sensitive to such PUFA effects, having a relatively poor capacity to both use and clear PUFAs.

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.

The significance of serum erythropoietin levels in assessing the severity of renal damage in children with reflux nephropathy.

PURPOSE: Erythropoietin (EPO) is the principal factor regulating red blood cell production in humans. It has been shown that EPO gradually decreases with the progression of diabetic nephropathy and may be used as a marker of severity of disease. In vitro studies have shown that interleukin-10 (IL-10) acts synergistically with EPO to increase stimulation of erythroid differentiation and proliferation. We evaluate serum levels of EPO and IL-10 in children with reflux nephropathy (RN). MATERIALS AND METHODS: Serum level of EPO and IL-10 were measured in 32 girls and 22 boys with RN, and in 22 boys and 10 girls who served as healthy controls. Renal scarring was evaluated with Technetium dimercapto-succinic acid scan. RN was severe (less than 20% uptake) in 16 children, moderate (20% to 40% uptake) in 25 and mild RN (greater than 40% uptake) in 13. Because anemia may further stimulate EPO production we also compared the index Hb (hemoglobin) x EPO in all patients. IL-10 and EPO were measured with standard enzyme-linked immunosorbent assay technique. The unpaired t test was used for statistical analysis. RESULTS: There were no statistically significant differences in the serum levels of EPO in children with RN (6.11 +/- 0.51 mIU/ml) compared to controls (6.42 +/- 0.46 mIU/ml) (p >0.5). Similarly the index Hb x EPO was 75.25 +/- 5.65 in children with RN compared to 73.76 +/- 5.48 in controls. Mean EPO levels were similar for mild, moderate and severe RN. There was no difference in the serum levels of IL-10 in children with RN (23.14 +/- 2.32 pg/ml) compared to controls (22.67 +/- 4.13 pg/ml) (p >0.5). CONCLUSIONS: Although EPO has been reported to be a marker of progressive renal disease in diabetic nephropathy, our data indicate that serum levels of EPO do not reflect the severity of renal parenchymal damage in children with RN.

HSP60 induces self-tolerance to repeated HSP60 stimulation and cross-tolerance to other pro-inflammatory stimuli.

In addition to their primary function as intracellular chaperone proteins, the immunomodulatory properties of heat shock proteins (HSP), including their role as adjuvants for vaccines, have become a focus of intense research interest. Interestingly, the effect of chronic exposure to an endogenous immunomodulator and initiator of inflammation such as autologous HSP60 has as yet remained uncharacterized. In this study, we demonstrate that pretreatment of monocytes with human HSP60 results in a suppression of TNF-alpha production on restimulation with HSP60. Furthermore, desensitization with HSP60 inhibits TNF-alpha expression in these cells in response to LPS stimulation, thereby inducing "cross-tolerance". In contrast to TNF-alpha suppression, IL-1beta expression was augmented in HSP60-pretreated monocytes on restimulation, while being suppressed in THP-1 cells. Addition of an anti-IL-10 neutralizing antibody had no significant effect on HSP60- or LPS-induced tolerance.HSP60 priming of monocytes also results in significant down-regulation of HLA-DR, CD86 and Toll-like receptor 4 expression, but minimally up-regulates CD80 expression, similar to that previously reported with LPS. By identifying a previously unrecognized "tolerizing" effect of extended exposure to autologous HSP60 on the innate immune system, as opposed to its recently identified pro-inflammatory stimulatory capacity, this study highlights a further level of complexity of our understanding of the biological activities of HSP.

Equivalent functional nuclear factor of activated T cell 1 mRNA and protein expression in cord blood and adult T cells.

Cord blood (CB) cells are now an important source of stem cells for bone marrow reconstitution as a result of the decreased risk of graft-versus-host disease that was attributed to previously reported absent or diminished nuclear factor of activated T (NFAT) cell 1 expression by CB T cells. This study reexamines NFAT1 expression in CB T cells. CB T cells were examined for NFAT1 mRNA and protein expression. These cells were then stimulated, and NFAT1 translocation and interleukin-2 production were assessed. Our results show that resting CB CD4+ T cells express NFAT1 mRNA and protein at levels similar to their adult counterparts. On stimulation, NFAT1 is translocated into the nucleus where DNA binding can occur, whereas calcineurin inhibition prevents interleukin-2 production. We conclude that differential responsiveness of CB T cells cannot be attributed to differences at the level of NFAT1 expression or its ability to function in these cells but may represent an altered sensitivity to stimulation.

IL-7-regulated homeostatic maintenance of recent thymic emigrants in association with caspase-mediated cell proliferation and apoptotic cell death.

Homeostasis of T cells is essential to the maintenance of the T cell pool and TCR diversity. In this study, mechanisms involved in the regulation of cytokine-mediated expansion of naive T cells in the absence of Ag, in particular the role of caspase activation and susceptibility to apoptosis of recent thymic emigrants (RTEs), were examined. Low level caspase-8 and caspase-3 activation was detected in proliferating IL-7-treated cells in the absence of cell death during the first days of culture. Caspase inhibitors suppressed IL-7-induced expansion of RTEs. Low level expression of CD95 and blocking Ab experiments indicated that this early caspase activation was CD95 independent. However, CD95 levels subsequently became dramatically up-regulated on proliferating naive T cells, and these cells became susceptible to CD95 ligation, resulting in high level caspase activation and apoptotic cell death. These results show a dual role for caspases in proliferation and in CD95-induced cell death during Ag-independent expansion of RTEs. This method of cell death in IL-7-expanded RTEs is a previously unrecognized mechanism for the homeostatic control of expanded T cells.

Nitrergic hyperinnervation in appendicitis and in appendices histologically classified as normal.

CONTEXT: The pathogenesis of appendicitis remains poorly understood. Despite new diagnostic techniques, appendices removed from patients with suspected appendicitis often appear histologically normal on conventional examination. There is increasing evidence of involvement of the enteric nervous system in immune regulation and in inflammatory responses in the gastrointestinal system. OBJECTIVE: To investigate the nitrergic innervation of (a) acutely inflamed appendices, (b) appendices classified as histologically normal from patients with a clinical diagnosis of appendicitis, and (c) normal control appendix specimens, using the whole-mount preparation technique. PATIENTS AND DESIGN: Full-thickness specimens were collected from 28 acutely inflamed appendices (age range, 3.2-13.4 years), 31 histologically normal appendices removed from patients (age range, 5.7-13.6 years) with suspected appendicitis, and 23 histologically normal appendices from patients (age range, newborn to 12.1 years) undergoing elective abdominal surgery (controls). Whole-mount preparation using nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and neuronal nitric oxide synthase immunohistochemistry were performed. The density of myenteric plexus was measured with a computerized analysis system. RESULTS: The density of myenteric plexus in normal appendix specimens was similar to that of large bowel from the newborn period up to 3 years of age; this density decreased significantly thereafter. The myenteric plexus of normal appendix specimens from patients older than 4 years demonstrated smaller ganglia connected by thin nerve bundles, compared to larger ganglia and nerve bundles in large bowel. Significant neuronal hypertrophy was found in 55% of acutely inflamed and 41% of histologically classified normal appendix specimens. The myenteric plexus of these appendix specimens had even thicker nerve bundles connecting an increased number of ganglion cells. CONCLUSIONS: Differences in the architecture of the myenteric plexus in patients older than 3 years suggest an altered function and motility of appendix in the early years of life. The significant increase in neuronal components of the myenteric plexus in a high proportion of acutely inflamed and histologically normal appendix specimens is unlikely to have developed during a single acute inflammatory episode. This suggests an underlying chronic abnormality as a secondary response to chronic luminal obstruction or repeated inflammatory episodes in the histologically normal appendix.

Up-regulation of angiotensin-converting enzyme (ACE) gene expression induces tubulointerstitial injury in reflux nephropathy.

Reflux nephropathy (RN) is the cause of end-stage renal failure in 3%-25% of children and 10%-15% of adults. Angiotensin-converting enzyme (ACE) converts the inactive decapeptide angiotensin I (Ang I) to the active octapeptide angiotensin II (Ang II), a potent vasoconstrictor. ACE is localized in highest concentrations on the luminal surface of endothelial cells, but is also found in several other cell types, including the epithelial cells of the proximal renal tubule. Recent studies have suggested that ACE increases production of the components of extracellular matrix (ECM) such as fibronectin (Fib) mediated through Ang II. Since RN is a primary tubulointerstitial disease, we hypothesized that local overexpression of ACE may induce renal fibrosis via up-regulation of Ang II. In this study, we investigated the expression of ACE in severely refluxing kidneys from eight patients (age range 6 months-14 years) with severe RN secondary to primary high-grade vesicoureteral reflux at nephrectomy. Control material included normal kidney specimens obtained from three adult patients during partial nephrectomy for an incidentaloma. Soluble enzyme immunohistochemistry was carried out using polyclonal antibodies to ACE and Fib. In-situ hybridization (ISH) was performed utilizing biotin-labelled antisense oligonucleotide probe. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the relative amount of ACE mRNA. In the refluxing kidney, there was strong ACE immunoreactivity in the glomerulus and proximal tubules and moderate-to-strong immunoreactivity in the distal tubules accompanied by strong Fib immunoreactivity in the glomerulus, proximal tubule, and interstitial space. There was strong ACE mRNA expression in the glomerulus and proximal tubules and moderate expression in the distal tubules. In the control kidneys, homogeneous weak ACE immunoreactivity and mRNA expression was demonstrated only in the proximal tubules. RT-PCR showed strong ACE expression in the refluxing kidneys compared to controls. Up-regulation of ACE in RN accompanied by an increase in ECM in the tubulointerstitial space suggests that ACE is involved in the pathogenesis of the renal parenchymal damage in patients with RN. Pharmacologic blockade of ACE may be helpful in preventing the renal fibrosis associated with RN.

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

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 gamma (IFN-gamma), 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-1beta 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-1beta 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.

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-beta production and only minimal TNF-alpha and IL-1beta 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-alpha and IL-1beta. This suppression of proinflammatory production was not reversible by depletion of IL-10 or TGF-beta or by addition of exogenous IFN-gamma. 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.