The kinetics and stimulant dependence of cytokine production by blood and bronchoalveolar lavage T cells evaluated at the single cell level.

We have previously shown that bronchoalveolar lavage (BAL) T cells from human airways predominantly produce interferon gamma (IFN-gamma) and interleukin 2 (IL-2) when stimulated ex vivo. The kinetics of TH1 and TH2 cell cytokine production by T cells from both blood and BAL were studied to establish the optimal time after stimulation either with pharbol myristate (PMA) and ionomycin or with the more physiological stimulus of anti-CD3 for intracellular cytokine detection of IFN-gamma, IL-2, IL-4 and IL-5 in both blood and BAL T cells. The optimal time for positive identification of IL-2 in both blood and BAL was 5 h after PMA/ionomycin stimulation, whereas the first peak for IFN-gamma was found after 5 h in blood but after only 3 h in BAL. T cells from different biological compartments responded differently to each of the stimuli. Whilst anti-CD3 stimulation did not induce TH1 cytokine production in blood T cells, it readily induced both IFN-gamma and IL-2 production in BAL T cells. The kinetics of cytokine production were found to be stimulus dependent. Whilst IL-2 production showed similar kinetics with both stimuli, the kinetics of IFN-gamma production differed between stimuli. We have also examined the effect of five different stimuli on cytokine production by T cells to determine whether different forms of stimulation may selectively stimulate or inhibit different cytokines. Not surprisingly, PMA/ionomycin induced a greater percentage of BAL T cells to produce TH1 cytokines. However, other than modest amounts of the TH2 cytokines IL-4 and IL-5 were not induced by any of the five stimuli.     

Spontaneous and stimulated interleukin-6 and tumor necrosis factor-alpha production at delivery and three months after birth

OBJECTIVE: To study the production and interrelations of maternal and neonatal cytokines (IL-6 and TNF-alpha) during labor, after vaginal delivery and at three months after delivery. METHOD: The unstimulated concentrations of cytokines in the supernatants of whole-blood cultures and concentrations after PMA (phorbol 12-myristate 13-acetate) and concanavalin (conA) stimulation were determined by enzyme-linked immunosorbent assays (ELISAs). The blood samples were from the peripheral veins of 27 healthy women during term labor and immediately after delivery and three months after delivery. Neonatal samples were taken at birth (cord blood) and three months after delivery. RESULTS: IL-6 responses to stimulation were increased in the parturients and in umbilical cord blood at delivery compared with maternal and neonatal samples obtained 3 months postpartum. In contrast, the production of maternal TNF-alpha in peripheral blood was down-regulated at delivery compared with values 3 months postpartum. After an IL-6 and TNF-alpha burst in umbilical cord samples, neonatal cytokine production was at a low level three months after delivery. IL-6 production tended to be higher in both umbilical cord blood as well as in maternal samples after delivery in women who were younger. In addition, TNF-alpha production in umbilical cord blood was significantly higher in those women who were younger. CONCLUSIONS: The production of IL-6 was up-regulated in both the maternal and in umbilical cord blood at delivery. The production of TNF-alpha was up-regulated in umbilical cord blood compared with neonatal values 3 months after birth. Maternal age had effects on IL-6 and TNF-alpha production at delivery.     

Kinetics of intracytoplasmic Th1 and Th2 cytokine production assessed by flow cytometry following in vitro activation of peripheral blood mononuclear cells

BACKGROUND: Standard cytokine detection methods are unable to determine which cells are the producing cells. We report on the extent and under which conditions the multilabeling capability of flow cytometry (FCM) can bring new advances into the field. METHODS: Five different cytokines, interleukin-2 (IL-2), -4, -5, -10 and interferon-gamma (IFN-gamma), were assessed simultaneously under five ex vivo stimulation conditions in peripheral blood mononuclear cells from five healthy volunteers in a 5-day kinetic study. A second group of 35 volunteers was assessed for IFN-gamma and IL-2 production. RESULTS: This study showed that (a) intracytoplasmic cytokines were almost undetectable within unstimulated cells, (b) intracytoplasmic cytokines were detected only in CD69(+) T lymphocytes, and (c) intracytoplasmic IL-2 and IFN-gamma were dramatically upregulated after stimulation with phorbol myristate acetate (PMA)-ionomycin in a biphasic response or with PMA-phytohemagglutinin (one major peak only at 18 h) but to a lesser extent with other stimuli such as monoclonal antibodies. Th2 cytokines were detected at a later time point and at lower levels. PMA/ionomycin stimulation after 4 h and 18 h of culture in 35 other volunteers individualized several subgroups according to the frequency of IFN-gamma- or IL-2-producing cells--IFN-gamma delayed producers (n = 10/35), IFN-gamma low producers (n = 8/35), and IL-2 delayed producers (n = 16/35)--as opposed to IFN-gamma or IL-2 normal producers. CONCLUSIONS: FCM appears to be a good tool to examine cell cytokine status in pathology (allergy, autoimmune disease, etc.) provided that optimal stimulation conditions and multiple time-point cultures are used. It also seems to be a relevant method to define new Th subsets further.     

Restricted T cell expression of IL-2/IFN-gamma mRNA in human inflammatory disease

It has been difficult to demonstrate functionally distinct T cell populations in humans on the basis of cytokine secretion. As previous investigators have examined the T cell cytokine profile from immunized animals, we examined whether Th1 or Th2 type T cells could be identified in the peripheral blood or cerebrospinal fluid (CSF) immune compartments from subjects with or without inflammatory diseases. Using limiting dilution analysis and growth with PHA and IL-2/IL-4, we directly cloned a total of 177 T cells from the peripheral blood and CSF of seven subjects, four with inflammatory disease and three control subjects, and examined the cytokine message profile after stimulation with ionomycin and PMA. We found that most clones from both the peripheral blood and CSF express IL-1, IL-2, IL-4, IFN-gamma, or TNF-alpha cytokine mRNA after activation with ionomycin and PMA. All T cell clones tested produced TNF-alpha mRNA, and all but 14 produced IFN-gamma mRNA. As reported previously, Th0 cells, which produced IFN-gamma, IL-2, IL-4, and IL-5 mRNA, were found in most subjects. In striking contrast, Th1 cells, which expressed IL-2 and IFN-gamma but not IL-4 or IL-5 mRNA, were present in both peripheral blood and CSF of subjects with inflammatory disease but not found in peripheral blood or CSF of subjects without systemic inflammation. Th2 cells, expressing IL-4 and IL-5 but not IFN-gamma or IL-2 mRNA, were not found in any subject. These data present the first evidence for Th1 T cell clones in humans that may be associated with systemic inflammation.     

Zinc modulates mRNA levels of cytokines

Zinc plays an important role in cell-mediated immune function. Altered cellular immune response resulting from zinc deficiency leads to frequent microbial infections, thymic atrophy, decreased natural killer activity, decreased thymic hormone activity, and altered cytokine production. In this study, we examined the effect of zinc deficiency on IL-2 and IFN-gamma in HUT-78 (Th0) and D1.1 (Th1) cell lines and TNF-alpha, IL-1 beta, and IL-8 in the HL-60 (monocyte-macrophage) cell line. The results demonstrate that zinc deficiency decreased the levels of IL-2 and IFN-gamma cytokines and mRNAs in HUT-78 after 6 h of PMA/p-phytohemagglutinin (PHA) stimulation and in D1.1 cells after 6 h of PHA/ionomycin stimulation compared with the zinc-sufficient cells. However, zinc deficiency increased the levels of TNF-alpha, IL-1 beta, and IL-8 cytokines and mRNAs in HL-60 cells after 6 h of PMA stimulation compared with zinc-sufficient cells. Actinomycin D study suggests that the changes in the levels of these cytokine mRNAs were not the result of the stability affected by zinc but might be the result of altered expression of these cytokine genes. These data demonstrate that zinc mediates positively the gene expression of IL-2 and IFN-gamma in the Th1 cell line and negatively TNF-alpha, IL-1 beta, and IL-8 in the monocyte-macrophage cell line. Our study shows that the effect of zinc on gene expression and production of cytokines is cell lineage specific.     

Flow cytometric visualisation of cytokine production by CD3-CD56+ NK cells and CD3+CD56+ NK-T cells in whole blood

BACKGROUND: Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring. METHODS: With a three-colour fluorescent labelling technique, specific cytokine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK population or on the CD3+/CD56+ NK-T-cell population. RESULTS: Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and ionomycin stimulation. CONCLUSIONS: This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3+CD56+ NK-T lymphocytes in patients with relevant diseases.     

Concentrations of progesterone in arterial and venous plasma of fetal pigs and their dams in late gestation

Blood samples were drawn from uterine arteries and veins of pregnant gilts and from the umbilical artery and vein of each of their fetuses during laparotomy at Day 80. Concentrations of progesterone (P) were greater in fetal than maternal plasma. Uptake of P from the placenta by the fetal blood was evident but was not equivalent to the maternal uterine arterial-venous difference in P concentration. No correlation between plasma P and fetal weight was noted. Concentrations of P in both umbilical vessels of female fetuses were higher than in male fetuses. These data indicate that fetal sex affects the rate of transport and/or synthesis of P in the utero/placental compartment and/or the rate of metabolism of P in the fetus. The relative importance of de novo synthesis and transplacental transport of P in establishing concentrations of P in fetal blood remains to be elucidated.     

Type 1 vs. type 2 cytokine secretion in vitro and its regulation by hydrocortisone in humans subjected to 120-day anti-orthostatic bed-rest regime

Head-Down Bed-Rest (HDBR) mimics some of the physiological stress effects of microgravity. Six healthy volunteers were subjected to bed-rest for 120 days. Blood samples were collected one month before (PRE), on day 110 of HDBR (DAY 110), and on the 7th day after bed-rest regime ends (POST). Distribution of T-cell subsets, NK-, B-cells and monocytes was assessed in the whole blood. Distribution of cytokine secreting T-cells was assessed in PMA/ionomycin cell culture. Peripheral Blood Mononuclear Cells (PBMC) and whole blood cells (WB) were activated with a combination of PHA and LPS to assess cytokine secretion. In addition, PHA/LPS activated cell cultures were treated with 10(-6) M of hydrocortisone (HCS) in order to study stress-induced alterations in the cortisol-sensitivity of immunocytes. Results from HCS culture were compared to non-treated control cultures. Stress factors of HDBR affect immune responsiveness and immune-endocrine homeostatic interrelations in vitro as follow: 1) alter expression of surface receptor to IL-2 (CD25) by CD4+ and CD8+ T-cell subsets in PHA/LPS activated PBMC culture; 2) alter distribution of IL-2 and/or IFN-gamma producing CD4+ and CD8+ T-cells in PMA/ionomycin activated culture; 3) significantly affect secretion of IL-2, IFN-gamma, and IL-4, but not IL-10 and soluble IL-2 receptor alpha in PHA/LPS activated PBMC culture; 4) shift Type 1 vs. Type 2 cytokine balance in PHA/LPS activated culture toward to Type 1 response; 5) in vitro treatment with hydrocortisone unequally modulate expression of CD25 on CD4+, and CD8+ T-cells, as well as secretion of Type 1 and Type 2 cytokines in PHA/LPS activated PBMC culture during bed-rest regime; 6) assessment of immune profile depends from the cellular and humoral milieu of cell culture.     

Subpopulations of fetal thymocytes defined by expression of T cell receptor/CD3 and IL-2 receptor. CD3 and IL-2 receptor alpha-chain are expressed on reciprocal cell populations

The expression of the TCR/CD3 complex and the IL-2R alpha chain (p55) on fetal thymocytes has been analyzed by flow cytometry (FCM). Two-parameter immunofluorescence identified three subpopulations which were respectively IL-2R alpha-/CD3+, IL-2R alpha+/CD3-, or IL-2R alpha-/CD3-; no detectable population of IL-2R alpha+/CD3+ cells was found in unstimulated fetal thymocytes. Fractionation by "panning" and by sterile flow cytometric separation was used to characterize the functional responsiveness of these three subpopulations to a variety of stimuli. All three populations proliferated in response to PMA + ionomycin + rIL-2. In contrast, stimulation with anti-CD3 + IL-2 induced proliferation in IL-2R alpha-/CD3+ and IL-2R alpha-/CD3- but not in IL-2R alpha+/CD3- thymocytes. IL-2R alpha- cells, including sorted IL-2R alpha-/CD3- thymocytes, underwent a phenotypic change in response to in vitro stimulation with anti-CD3 + IL-2, resulting in the appearance of an IL-2R alpha+/CD3+ population that was not detected in freshly isolated thymocytes. The ability of fractionated fetal thymocytes to produce lymphokine in response to PMA + ionomycin was also evaluated. Only the IL-2R alpha-/CD3- fraction generated detectable IL-2. These findings demonstrate for the first time that CD3 and IL-2R alpha are expressed in a mutually exclusive fashion in fetal thymocytes and define three subpopulations of thymocytes that differ significantly in their proliferative and differentiative responses to TCR-mediated, IL-2R-mediated, and pharmacologic stimulation.     

CD4+ and γδTCR+ T lymphocytes are sources of interleukin-17 in swine

In the veterinary field, only limited information is available about interleukin-17A (IL-17), despite the fact that this cytokine plays an important role during pro-inflammatory immune responses and induces the production of chemotactic factors for neutrophils. The aim of this study was to characterize porcine IL-17-producing cells. We tested the cross-reactivity of five anti-human IL-17 monoclonal antibodies because such antibodies against porcine IL-17 are currently unavailable. Whole blood cells (WBCs) were stimulated with phorbol-myristate-acetate (PMA) and ionomycin and subsequently analyzed by flow cytometry. The antibody clone SCPL1362 was found to cross-react with porcine IL-17, whereas the other four antibodies tested did not recognize this cytokine. Using this antibody, we characterized porcine WBC-secreting IL-17 after PMA and ionomycin stimulation. All IL-17-producing WBCs were positive for the T lymphocyte marker CD3. Myeloid cells (CD172α(+)) and B lymphocytes (CD79α(+)) were IL-17 negative. The major subset of IL-17 positive T lymphocytes was the CD4(+) lymphocytes (about 60% of all IL-17 positive WBCs). The remaining IL-17 positive WBCs were γδTCR(+) lymphocytes. CD8 positive and CD8 negative cells were found within both CD4(+) and γδTCR(+) cells producing the cytokine. Moreover, IL-17 positive cells were mostly CD45RA negative, therefore activated cells or memory cells. Flow cytometry data were confirmed using sorted cells. Both sorted CD4(+) and γδTCR(+) cells produced IL-17 at mRNA level after PMA and ionomycin stimulation while double negative CD4(-)γδTCR(-) cells were negative for IL-17. We can conclude that only two subpopulations of porcine WBCs are sources of IL-17 after non-specific stimulation: CD3(+)CD4(+) and CD3(+)γδTCR(+).     

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles,a novel class of immunomodulators

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.     

Elevation of plasma 7B2 (a novel pituitary protein) in cord blood at obstetrical delivery and the possible correlation with GH

Plasma 7B2-immunoreactivity (7B2-IR) concentrations in umbilical artery (UA), umbilical vein (UV) and maternal vein (MV) were measured by RIA at the time of obstetrical delivery at term. Plasma 7B2-IR concentrations (Mean +/- SEM) in UA (N = 12), UV (N = 16) and MV (N = 16) were 725 +/- 69, 699 +/- 64 and 116 +/- 4.5 pg/ml, respectively. Plasma 7B2-IR concentrations in UA and UV were much higher than those in MV. There was no arterio-venous gradient between UA and UV. A trace amount of 7B2-IR (Mean +/- SEM, 226 +/- 16.8 pg/g tissue) was detected in the placental extracts. A statistically significant positive correlation (r = 0.7595, p less than 0.005) was found between plasma 7B2-IR and GH in the UV. Significant negative correlations between body weight of the neonates and plasma levels of GH (r = -0.6836, p less than 0.01) and 7B2-IR (r = -0.4939, p less than 0.05) were also apparent. When analyzing cord blood plasma using gel permeation chromatography and SDS-polyacrylamide gel electrophoresis, a major peak with an apparent molecular weight of 20,000 was observed. These findings suggest that 7B2-IR in UA and UV originates from the fetus and that 7B2-IR does not permeate through the placenta. The possibility of involvement of 7B2 in fetal growth warrants attention.     

Intracellular T-cell cytokine levels are age-dependent in healthy children and adults

Intracellular detection of cytokines via fluorescent antibody staining and flow cytometry has quickly become a standard method in experimental immunology. However, in pediatrics most studies have been hampered by the exclusion of healthy control individuals or have been skewed by neglecting to observe age-dependent differences in cytokine production. We therefore intended to establish normal values for different age groups and to describe the age-dependent development of cytokine profiles. Whole blood from 46 healthy children and 33 adults was analyzed by flow cytometry after stimulation with PMA, ionomycin and Mmonensin, and staining with anti-cytokine and surface antibodies. In the pediatric population, we found a significant positive correlation between age and intracellular cytokine levels of IFN-gamma, IL-2, IL-4 and TNF-alpha in CD4+ cells, as well as for IFN-gamma and TNF-alpha in CD8+ cells. In adulthood, no such striking trend could be detected, but significant correlation was found for IL-10 in CD4+ cells and IFN-gamma in CD8+ cells as well as for TNF-alpha in both cell subgroups. We present here the first systematic analysis of intracellular cytokine production in normal, healthy children between the ages of 0 to 18 years compared to results in adults. These data may provide a reference basis for the study of cytokine secretion patterns, and they also demonstrate a significant maturation of the T-cell cytokine production capacity from birth to adulthood.     

Increased specific T cell cytokine responses in patients with active pulmonary tuberculosis from Central Africa

An understanding of T cell responses that are crucial for control of Mycobacterium tuberculosis (MTB) has major implications for the development of immune-based interventions. We studied the frequency of purified protein derivative (PPD)-specific CD3) cells expressing interleukin-2 (IL)-2, gamma interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and IL-10 in HIV-negative pulmonary tuberculosis patients (TB, n=30) as well as in healthy individuals (controls, n=21) from Central Africa. Increased frequencies of PPD-stimulated CD3+ cells expressing IL-2, IFN-gamma, and TNF-alpha in TB were seen when compared with frequencies of controls. The presence of type 1 cytokine biased responses in TB patients was supported by a shift in the distribution pattern of cytokine expression from exclusively IL-2 or TNF-alpha expression seen in controls towards an increased frequency of IFN-gamma/IL-2 or IFN-gamma/TNF-alpha co-expression in TB. Higher levels of PPD-induced IFN-gamma in the supernatants from TB patients than from controls were found, which correlated with its intracellular expression. PPD was a weak inducer of IL-10 in T cells and insufficient in promoting cytokine production in TCRgammadelta+CD3+ cells. Non-specific stimulation with PMA and ionomycin revealed increased frequencies of CD4+ cells expressing IFN-gamma in controls, while expression of IL-2, IL-4, IL-10, IL-13, and TNF-alpha was not different. Non-specific cytokine responses of TCRgammadelta+CD3+ cells were similar in all groups. Pulmonary TB in Central Africa is associated with enhanced expression and secretion of specifically induced cytokines that are frequently implicated in host defense against MTB.     

Proinflammatory cytokines in bovine colostrum potentiate the mitogenic response of peripheral blood mononuclear cells from newborn calves through IL-2 and CD25 expression

Abstract: Bovine colostrum contains high concentrations of cytokines, and colostral cytokines are considered to be an important factor in stimulation of maturation of the immune system in newborns. In this study, 5 proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma and IL-1 receptor antagonist, IL-1ra) present in colostrum were tested for their potential to enhance mitogenic response and to elicit expression of IL-2 mRNA and CD25 in peripheral blood mononuclear cells (PBMC) from newborn calves before being fed colostrum. PBMC were pretreated with each recombinant bovine cytokine for 2 hr before stimulation with concanavalin A (ConA). Pretreatment of PBMC from newborn calves with IL-1beta, TNF-alpha or IFN-gamma significantly enhanced the ConA response, whereas IL-1ra inhibited the response. The degree of enhancement or inhibition of mitogenic response by these cytokines was more pronounced in PBMC from newborn calves than in those from adult cows. Although IL-2 mRNA expression in ConA-stimulated PBMC from newborn calves was weaker than that in those from adult cows of ConA-stimulated controls, the expression levels became comparable after pretreatment with IL-1beta, TNF-alpha or IFN-gamma. The CD25 expression in PBMC from newborn calves was also enhanced by pretreatment with IL-1beta, TNF-beta and IFN-gamma. These results suggest that pretreatment of neonatal PBMC with IL-1beta, TNF-alpha or IFN-gamma promotes mitogenic response to ConA through up-regulating the production of IL-2 and the expression of the mature IL-2 receptor.     

Expression of IL-17 in human memory CD45RO+ T lymphocytes and its regulation by protein kinase A pathway.

In the present study, the authors compared the interleukin 17 (IL-17 expression of human naive and phenotypically defined memory T cells as well as its regulation by cAMP pathway. Our data showed that IL-17 mRNA was highly expressed in memory human peripheral CD8(+)45RO+T cells and CD4(+)45RO+T cells when peripheral blood mononuclear cells were first stimulated with ionomycin/PMA. IL-17 expression in memory CD8(+)T cells required accessory signals since culture of ionomycin/PMA-activated CD8(+)45RO+T cells alone did not result to IL-17 expression. In contrast, memory CD4(+)T cell population seems to be more independent. IL-17 and interferon gamma(IFN-gamma) mRNA were both inhibited in the presence of PGE2 or the cAMP analogue (dibutyryl-cAMP), while the anti-inflammatory cytokine IL-10 was highly increased. In contrast, naive CD45RA+T cells were unable to express IL-17 whatever the culture conditions. Naive CD4(+)and CD8(+)T cells were sensitive to the PKA regulatory pathway since they represent a significant source of IL-10 when PBMC were first cultured with ionomycin/PMA in the presence of either PGE2 or db-cAMP. The authors showed that naive cells are highly dependent to their microenvironment, since culture of ionomycin/PMA-activated CD45RA+T cells alone did not result in detectable levels of cytokines even in the presence of PGE2. Results also showed that PGE2 induced quite the same levels of intracellular cAMP in naive and memory cells suggesting that these cell populations are equally sensitive to PGE2. However, we suggest that PGE2 may be more efficient in blocking both IL-17 and IFN-gamma expression in already primed memory T cells, rather than in suppressing naive T cells that could represent a significant source of IL-10. Data suggest that PKA activation pathway plays a critical role in the regulation of cytokine profiles and consequently the functional properties of both human naive and memory CD4(+) and CD8(+)T cells during the immune and inflammatory processes.     

Agents that mimic antigen receptor signaling inhibit proliferation of cloned murine T lymphocytes induced by IL-2

We have shown previously that stimulation of cloned murine T lymphocytes via the TCR inhibits their responsiveness to rIL-2. Signaling via the TCR is believed to result in a variety of biochemical events that include a rise in intracellular free calcium and activation (translocation) of protein kinase C. These two signals also can be generated by calcium ionophores, such as ionomycin, and by activators of protein kinase C, such as PMA. We report here that treatment of cloned murine T lymphocytes with PMA, ionomycin, or the combination led to a dose-dependent inhibition of IL-2-dependent proliferation but did not inhibit lymphokine secretion. Concentrations of PMA and ionomycin that maximally inhibited proliferation stimulated maximal lymphokine secretion and increased mitochondrial activity as assessed by measurement of cleavage of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium-bromide. Furthermore, PMA, ionomycin, the combination, or immobilized anti-CD3 mAb added after 12 to 16 h of culture with IL-2 could inhibit proliferation. These results demonstrate that PMA and ionomycin mimic stimulation of the TCR by high concentrations of immobilized anti-TCR mAb in that proliferation is inhibited and lymphokine secretion is induced. In addition, PMA or ionomycin could independently inhibit proliferation of some cells. These findings suggest that alternative mechanisms exist to regulate proliferation. Either increased levels of intracellular calcium or the physiologic events corresponding to those induced by PMA can inhibit IL-2-dependent replication of T lymphocytes.     

The timed-pregnant baboon animal model can be used for determining the role of soluble vascular endothelial growth factor receptors 1 and 2 during development

BACKGROUND: During pregnancy, mechanisms that allow for regulation of continuous fetal and placental vasculogenesis with prevention of maternal neo-vascularization remain elusive. The vascular endothelial growth factor (VEGF) biological system has a key role during vasculogenesis. The aims of this study were to validate a bioassay for soluble vascular endothelial growth factor receptors 1 and 2 (sVEGFR-1 and sVEGFR-2) in baboon plasma and to determine the maternal and fetal plasma concentration of these receptors at the end of the baboon pregnancy. METHODS: Maternal peripheral blood samples were obtained from eight baboons (Papio anubis) prior to elective cesarean section and from the umbilical cord after the fetuses were delivered. Spike and recovery experiments at various concentrations in pooled baboon maternal plasma were used to validate a human quantitative sandwich immunoassay for sVEGFR-1 and -2. Concentrations of sVEGFR-1 and -2 were then determined in maternal and fetal plasma samples. RESULTS: No significant correlations were observed between sVEGFR-1 or -2 concentrations in maternal and fetal circulations. The concentration of sVEGFR-1 was at least 30 times greater and that of sVEGFR-2 approximately two times greater, in maternal than in cord plasma (both P < 0.01). CONCLUSION: These findings suggest that baboons can be used to study the regulation of vasculogenesis during pregnancy.