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 gamma-chain cytokines, in particular IL-7, they show enhanced potential to survive, entry into cell cycle, and proliferation. Although common gamma-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.

Polyunsaturated fatty acids influence neonatal monocyte survival.

The n-3 and n-6 polyunsaturated fatty acids (PUFAs) are essential dietary constituents. They are potent modulators of the human immune response, and research has endeavoured to optimise the ratio of n-3 to n-6 fatty acids in the lipid emulsion component of total parenteral nutrition to harness their beneficial effects in the clinical setting. PUFAs modulate apoptosis of certain tumour cells and cell lines. Monocytes, which are major effector cells of the innate immune system, play a central role in the initiation, development, and outcome of the immune response. They are crucial in the defence against invading pathogens and are involved in the lysis of infected or malignant cells, wound healing, repair, and remodeling of tissues. In the present study we investigated whether PUFAs might evoke apoptosis in newborn monocytes. Purified cord-blood monocytes collected from uncomplicated full-term pregnancies were incubated for 24 h in complete medium in the presence or absence of one of the n-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic (EPA) or the n-6 PUFA arachidonic acid (AA). Following incubation, cells were triple-labelled with annexin V, CD14, and propidium iodide prior to flow-cytometric analysis to determine the degree of cell death. All experiments were performed in triplicate and data expressed as mean +/- 1 S.D. (%). In the absence of fatty acids, 30 +/- 4% of control cord monocytes underwent apoptosis or necrosis after 24 h incubation. At a concentration of 50 microM, none of the PUFAs had a significant effect on monocyte cell death, but at a dose of 100 microM, DHA resulted in 60 +/- 4% cell death (P < 0.05) while the other PUFAs had no significant effect. In contrast, at higher concentrations (200 microM), all the PUFAs significantly increased monocyte cell death (AA: 70 +/- 5%, DHA: 86 +/- 2%, EPA: 70 +/- 4%). PUFAs thus exert a potent influence on cord monocyte cell survival in vitro. Their effect is dose-dependent and DHA appears to be the most potent of the fatty acids tested. The influence of PUFAs on neonatal monocyte-cell survival suggests a novel mechanism whereby PUFAs may modulate the immune response.

Evidence of an inflammatory pathologic condition in "normal" appendices following emergency appendectomy.

BACKGROUND: Appendices removed from patients with suspected appendicitis often appear normal on histologic examination. OBJECTIVE: To study appendix specimens for the expression of inflammatory markers as an indicator of presence of an inflammatory response in this subgroup of patients. METHODS: Cyclooxygenase 1 and 2, prostaglandin E(2), inducible nitric oxide synthase, and major histocompatibility complex class II were investigated by immunofluorohistochemistry using confocal laser microscopy in 15 acutely inflamed appendix specimens, 39 histologically classified "normal" appendices, and 11 negative control specimens. RESULTS: Strong expressions of all the inflammatory mediators were found in the mucosa of inflamed appendices, in approximately 50% of histologically normal appendices from patients with a clinical diagnosis of appendicitis, and in none of the normal control specimens. CONCLUSION: This study confirms the existence of a subgroup of appendicitis within the so-called histologically normal appendices in which evidence of an inflammatory pathologic condition is only obvious at a molecular level. The initiating signal for this and all other forms of clinical appendicitis still remains elusive.

Neuronal hypertrophy in acute appendicitis.

OBJECTIVE: The pathogenesis of appendicitis remains poorly understood. However, there is increasing evidence of involvement of the enteric nervous system in immune regulation and in inflammatory responses. This study was set up to characterize the status of the enteric nervous system in normal and in inflamed appendixes. METHODS: S100- and 2',2'-cyclic nucleotide 3' phosphodiesterase-positive Schwann cells, synaptophysin, and neuron-specific, enolase-positive nerve fibers and tryptase-positive mast cells were evaluated with immunohistochemical staining in surgically resected appendixes from 20 children with histologically proven acute appendicitis (HA), 10 histologically normal appendixes (HN) from patients with a clinical diagnosis of appendicitis, and 10 normal appendixes from patients undergoing elective abdominal surgery. Immunostained sections were subjected to quantitative image analysis. The number and size of ganglia and the number of nerve fibers, Schwann cells, and mast cells in each tissue compartment was quantitatively or semiquantitatively measured. RESULTS: Increased numbers of fibers, Schwann cells, and enlarged ganglia, widely distributed in the muscularis externa and submucosa, were seen in all HA appendixes and in 4 of 10 HN appendixes. The number and size of ganglia in muscularis externa and in the submucosa of appendixes with HA were significantly greater compared with those in control appendixes (P <.001). A significantly increased number of individually stained nerve fibers and Schwann cells (P <.05) were present in the muscularis externa in HA appendixes compared with control appendixes. Significantly increased numbers of tryptase-positive mast cells (P <.05) were present in the submucosa, muscularis, and especially in the lamina propria in HA specimens, compared with that of control tissue. CONCLUSIONS: The significant increase in neural components and mast cells in acute appendicitis is unlikely to develop during a single acute inflammatory episode. This suggests an underlying chronic abnormality as a secondary reaction to repeated bouts of inflammation, obstruction, or both. These results challenge our current understanding of the pathophysiological processes that give rise to acute appendicitis.

Rapid conversion of naive to effector T cell function counteracts diminished primary human newborn T cell responses.

The reduced incidence of graft versus host disease following the use of human cord blood as a source of stem cells for bone marrow reconstitution challenges our understanding of the immunocompetence of newborn T cells. Newborn CD4+ T cells express mainly the CD45RA phenotype and have been considered to respond comparably to adult CD4+ T cells exhibiting the CD45RA phenotype. We compared the in vitro kinetics of phenotypic conversion of newborn and adult CD4+CD45RA+ T cells to CD4+CD45RO+ T cells. The cytokine profile and B cell helper activity of the converted CD4+CD45RO+ T cell population were also determined. Newborn CD4+CD45RA+ T cells were converted to CD4+CD45RO+ with significantly faster time kinetics than adult CD4+CD45RA+ T cells, following either phytohaemagglutinin (PHA) or anti-CD2 activation. Freshly purified newborn naive T cells did not produce IL-2, IL-4 or interferon-gamma (IFN-gamma) following stimulation, whereas adult naive T cells secreted IL-2 and adult-derived CD4+CD45RO+ T cells secreted all three cytokines under the same stimulatory conditions. However, newborn and adult CD4+CD45RA+ T cells, following primary stimulation and maturation in vitro, acquired the ability to secrete a Th1-type cytokine profile of IL-2 and IFN-gamma after secondary stimulation. Newborn CD4+ naive T cells that acquired the CD45RO phenotype in vitro also gained B cell helper activity equivalent to that of adult in vitro matured CD4+ naive T cells. These findings suggest that newborn and adult CD4+CD45RA+ T cell subsets are differentially responsive to various stimuli. They show that newborn CD4+CD45RA+ naive T cells can transform more quickly than their adult counterparts into functionally equivalent CD4+CD45RO+ T cells, a process that may be important to counteract the immature immune environment which exists in the newborn.

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-gamma-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.

Phosphorylation and the actin cytoskeleton in defective newborn neutrophil chemotaxis.

We have investigated the role of actin polymerization in the defective polymorphonuclear neutrophil (PMN) chemotaxis of the human newborn, and its regulation by protein kinase C and by phosphatases 1 and 2A. Isolated PMNs from adult volunteers and healthy term newborns, i.e. umbilical cord blood, were studied. Chemotaxis was measured by a modified micropore filter assay, and actin polymerization was assessed by flow cytometry. Chemotaxis of newborn PMNs (median 18 microm, range 9-21 microm) was significantly reduced compared with adult PMNs (median 23 microm, range 17-34 microm) (p < 0.001). Coincubation with the protein kinase C inhibitor bisindolylmaleimide GF109203X, did not significantly alter chemotaxis, whereas coincubation with the phosphatase inhibitors calyculin A or okadaic acid caused parallel dose-dependent inhibition of chemotaxis in adult and newborn PMNs. Peak actin polymerization was reduced in newborn compared with adult PMNs in response to stimulation with formyl-methionyl-leucyl-phenylalanine and zymosan-activated serum, but was normal in response to phorbol myristate acetate. Prior incubation for 5 min with bisindolylmaleimide GF109203X, calyculin A, or okadaic acid caused no significant alterations in the actin polymerization response to stimulation with formyl-methionyl-leucyl-phenylalanine. We conclude that: 1) newborn PMNs have reduced actin polymerization in response to stimulation with chemotactic agents which act via cell surface receptors, but not with phorbol myristate acetate, which acts directly in the cytoplasm. This suggests that a defect in cell signal transduction may be an underlying factor in defective newborn PMN chemotaxis. 2) Phosphatase inhibitors strongly inhibit chemotaxis but not actin polymerization, therefore phosphatases 1 and 2A may be important regulators of PMN chemotaxis, but this regulation takes place either at a point distal to actin polymerization or via another pathway. 3) Similar results in adult and newborn PMNs suggest that this is not the site of the underlying defect in newborn PMN chemotaxis.

Cord blood 'naive' T cells demonstrate distinct immunological properties compared with their adult counterparts.

Distinct immunological properties of naive T cells in cord blood as compared to their adult counterparts are likely to have consequences in the outcome of cord blood transplants.

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity.

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.

Differential cytotoxicity of cord blood and bone marrow-derived natural killer cells.

Allogeneic cord blood is now being widely used as a source of stem cells for hematologic reconstitution after myeloablative therapy, with reported significantly lower levels of graft-versus-host disease (GVHD) compared with the use of allogeneic bone marrow (BM). This study was undertaken to investigate biologic aspects of natural killer (NK) cell activity, as recognized effector cells of the GVHD and graft-versus-leukemia (GVL) response, from cord blood and conventional BM. NK-cell activity levels of freshly isolated cells from cord blood and BM against K562 targets were comparable. Lymphokine activated killer (LAK) cells from both hematopoietic cell sources were compared for their ability to kill target cells by necrotic or apoptotic mechanisms using specific target cell lines. Cord blood cells had significantly higher necrosis-mediated cytotoxic activity against Daudi target cells compared with BM-derived cells. Cord blood LAK cells had relatively high levels of apoptotic-mediated cytotoxicity against YAC-1 target cells, whereas BM-derived LAK cells were unable to induce apoptosis in these cells. Interleukin-2 (IL-2) induced significant granzyme B activity in cord cells in contrast to BM cells, in which very little activity was measured. Western blotting confirmed these findings, with IL-2 inducing granzyme B protein expression in cord cells but not detectable levels in BM cells. BM cells had significantly lower cell surface expression of IL-2R and prolonged culture in IL-2 was only partially able to restore their deficient apoptotic cytotoxic activity. Thus, major differences exist between cord blood-derived and BM-derived mononuclear cells with respect to their NK-cell-associated cytotoxic behavior. This could have important implications for stem cell transplantation phenomena, because it suggests that cord blood may have increased potential for a GVL effect.

Effect of major surgery on neutrophil chemotaxis and actin polymerization in neonates and children.

The authors have examined the effect of major surgery in neonates and older children on neutrophil (PMN) chemotaxis and on actin polymerization, an essential early step in PMN movement. Isolated PMNs from the following subjects were studied: healthy adult volunteers (n = 28), healthy newborns (n = 21), newborns undergoing major surgery (n = 7), and older infants and children undergoing major surgery (n = 14). Chemotaxis was measured by a micropore filter assay, and actin polymerization was measured by flow cytometry. Blood samples from surgical patients were obtained preoperatively, hourly during the procedure, immediately postoperatively, and 48 hours after surgery. Mean preoperative newborn PMN chemotaxis was similar to that of healthy newborn PMN, and mean preoperative PMN chemotaxis in children was similar to that of healthy adults. There were no significant alterations in PMN chemotaxis during or after major surgery in neonates or children. Peak PMN actin polymerization, after stimulation with formyl methionyl leucyl phenylalanine (FMLP) (10 nm), was significantly diminished in healthy neonates compared with adults (P < .005). Preoperative surgical neonates had similar peak PMN actin polymerization levels to those of healthy newborns, and older preoperative children had similar levels to adults. PMN actin polymerization did not significantly change during or after major surgery. Despite reductions in PMN chemotaxis and actin polymerization in healthy neonates, there is no further impairment of these PMN functions during or after major surgery. Our data suggest that PMN chemotactic function is resistant to the stress of uncomplicated major surgery in neonates and children.

Cord blood CD4+ CD45RA+ T cells achieve a lower magnitude of activation when compared with their adult counterparts.

Highly purified CD4+ CD45RA+ cells from cord blood and peripheral blood from healthy adults were studied. The levels of expression of the CD2, CD3, CD4 and CD28 antigens were similar; however, CD45 and CD45RA antigen expression were slightly lower in cord cells. The reduced expression of the CD45RA antigen on cord CD4+ T cells was confirmed in whole blood. Functional assessment revealed deficiencies in cord CD4+ CD45RA+ T cells. Interleukin-2 (IL-2) production in response to specific triggering via CD2 monoclonal antibody (mAb) alone, or CD2 mAb in combination with CD28 mAb showed marked underproduction (about 10% of adult production). When CD25 expression was examined, it was observed that the proportion of activated CD4+ CD45RA+ T cells in cord blood was lower than in adult (about 20% of adult expression). Proliferation to CD2 mAbs or CD2 + 28 mAbs of cord blood native cells was similarly depressed. Investigation of IL-2 mRNA expression under these stimulatory conditions paralleled the results observed for CD25 expression, IL-2 production and proliferation. When phorbol 12-myristate 13-acetate (PMA) was added to the cells triggered with CD2 + 28mAbs, the responses examined were enhanced in both cord and adult blood with no significant differences between the groups. These findings suggest that under identical conditions of stimulation, purified cord blood CD4+ CD45RA+ T cells do not acquire similar activation status as their adult counterparts. These findings may help in understanding the reduced graft-versus-host disease (GVHD) observed in cord blood stem cell transplantation.

CD72 ligation regulates defective naive newborn B cell responses.

The biological basis for reduced Ig production by naive newborn B cells compared to adult peripheral blood B cells is not fully understood. In a Con A + IL-2 T cell-dependent system using "competent" adult T cells, adult B cells produced large amounts of IgM, IgG, and IgA, while cord B cells were restricted to low levels of only IgM production. Cord B cell activation was also diminished. The contribution of specific B-T cell contact-mediated events to the diminished cord B cell response in this system, using mAbs to CD40, CD28, CD80, and CD72, were investigated, as well as regulation of B cell Ig production by cytokines. alphaCD72 ligation increased cord B cell activation and IgM production, but did not affect adult B cells. Blocking alphaCD40 mAb inhibited cord B cell Ig production completely, but only partly inhibited adult B cell Ig production even at high concentration, suggesting a greater sensitivity of cord B cells to disruption of the CD40-CD40L interaction. Addition of IL-10 did not increase cord B cell Ig production, while adult B cell Ig production was increased. However, combined addition of IL-10 and alphaCD72 significantly increased cord B cell Ig production over that in the presence of either alphaCD72 or IL-10 alone, but had no effect on adult B cells over that of IL-10 alone. These data suggest that the diminished T cell-dependent response of cord B cells is due to reduced or absent CD72 ligation. CD72 ligation plays an important role in the induction of primary responses by naive B cells. CD72 modulation of naive B cell sensitivity to IL-10 stimulation may have implications in the induction of class switch, which is deficient in newborn B cells. Since all T cells express CD5 constitutively, these data also suggest the existence of another ligand for CD72.

Linkage of protein kinase C-beta activation and intracellular interleukin-2 accumulation in human naive CD4 T cells.

A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long-term cellular functions. Protein kinase C-beta (PKC-beta) has been reported to be involved in interleukin-2 (IL-2) synthesis in T lymphocytes. In this study, the role of PKC-beta in intracellular accumulation of IL-2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (alpha and beta I) Go6976, and one which inhibits both classical and non-classical isotypes, GF109203X, caused a complete block in cytoplasmic IL-2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL-2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC-beta translocation. Flow cytometric assessments of PKC-beta and its isoforms beta I and beta II correlated with Western blotting analysis and these results were further supported by the use of PKC-beta-positive (HUT 78) and -negative (BW5147) T-cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC-beta is critical for accumulation of intracellular IL-2. The influence of CsA in reducing but not blocking IL-2 synthesis is discussed. PMA-induced down-regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non-classical PKC isoforms.

Differential expression of function-related antigens on newborn and adult monocyte subpopulations.

Umbilical cord blood and adult peripheral blood monocytes were separated into two subpopulations, based on the intensity of CD14 expression, and the coexpression of various antigens associated with monocyte function was examined. The majority of cord and adult monocytes expressed CD14 at a high density (CD14bright) while approximately 15% of monocytes expressed this antigen at a lower level (CD14dim). Three times as many CD14dim monocytes expressed CD16 (Fc gamma RIII) as did CD14bright monocytes in both cord and adult preparations, while its level of expression (mean fluorescence intensity) was significantly reduced on cord CD14dim monocytes relative to adult CD14dim monocytes. The percentage of cord blood CD14dim monocytes expressing human leucocyte antigen-DR (major histocompatibility complex class II antigen) was significantly reduced relative to adult CD14dim cells; however, the level of expression of this antigen was greater on both cord and adult CD14dim monocytes compared with CD14bright cells. Cord CD14bright cells expressed CD36 (OKM5) to a greater extent than did their adult counterparts. The level of expression of CD62L was reduced on cord CD14dim cells compared with adult CD14dim cells. CD11b (CR3) was expressed both on a higher percentage of cells and also with a greater intensity on both cord and adult CD14bright monocytes compared with their CD14dim counterpart. These results show that significant differences exist between cord and adult monocyte subpopulations with regard to expression of various antigens associated with specific effector function.

Antigen-independent responsiveness to interleukin-4 demonstrates differential regulation of newborn human T cells.

The low incidence of graft-versus-host disease following clinical use of umbilical cord blood compared to adult bone marrow as a source of stem cells for bone marrow reconstitution, leads to questions concerning the level of immunocompetence of newborn T cells. The maturation and functional status of newborn CD4+ T cells, which are almost exclusively CD45RA+ naive T cells, compared with their adult phenotypic counterparts, is poorly understood. We examined the proliferative response to mitogens and cytokines of CD4/CD45RA+ T cells from adults and newborns, with and without accessory cells. Newborn CD4/CD45RA+ T cells demonstrated a distinct proliferative response profile which was determined by the number of accessory cells present in co-cultures with various stimuli. Newborn CD4/CD45RA+ T cells were particularly responsive to interleukin (IL)-4, IL-4 plus anti-CD2 monoclonal antibodies (mAb) and IL-4 plus phytohemagglutinin (PHA), whereas adult CD4/CD45RA+ T cells were unresponsive under similar conditions. The mitogenic responses of newborn and adult CD4/CD45RA+ T cells to PHA and anti-CD2 mAb, which were equivalent, were directly proportional to the number of accessory cells present, whereas the responsiveness to cytokines was inversely proportional to the number of co-cultured accessory cells. Anti-CD2 responses were much more sensitive to low numbers of accessory cells than PHA. The particular sensitivity of newborn CD4/CD45RA+ T cells to IL-4 represents an antigen-independent T cell activation response which could help promote a Th2 immune response resulting in the newborn.