Effects of RRR-alpha-tocopherol on leukocyte expression of HSP72 in response to exhaustive treadmill exercise.

Previous research revealed an increased expression of HSP72 in leukocytes after vigorous endurance exercise. We questioned whether more intensive but shorter exercise also induces leukocyte HSP72 synthesis. To delineate the role of reactive oxygen species (ROS) in exercise-related HSP72 induction, we additionally examined the effect of RRR-alpha-tocopherol (alpha-toc) on HSP72 expression using a double-blind placebo (P) controlled cross-over design. After supplementation with alpha-toc (500 I.U. daily) or P for 8 days, 9 male subjects performed a combined exhaustive treadmill protocol (total duration 29.4 +/- 2.0 min). HSP72 was assessed on mRNA (RT-PCR) and protein levels (flow cytometry). HSP72 mRNA rose 3 h after exercise only in the P group, but individual differences (alpha-toc - P) did not reveal significant treatment effects. A moderate but significant rise of HSP72 protein occurred in granulocytes up to 48 h after exercise. Three hours post-exercise, granulocyte HSP72 protein was lower when subjects received alpha-toc, but this effect vanished 24 and 48 h post-exercise. Exhaustive treadmill exercise augments HSP72 mRNA in leukocytes and induced a moderate but prolonged response of granulocyte HSP72 protein. These exercise effects are lower when compared to earlier findings obtained after vigorous endurance exercise. ROS seem to be involved, but do not play the major role in the induction of granulocyte HSP72 synthesis after exhaustive exercise.

Basal expression of leukocyte iNOS-mRNA is attenuated in moderately endurance-trained subjects.

Nitric oxide (NO) is generated in immunocompetent cells by inducible NO-synthase (iNOS), and plays an important role in host defense. However, when produced in large amounts, NO can also exert damaging effects, a scenario that is observed during several inflammatory processes. In the study presented here, we investigated the impact of moderate endurance training (running volume: mean 53.1 km x week(-1), 95% confidence interval 41.2-65.1 km week(-1)) on the leukocyte expression of iNOS mRNA. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was used to examine iNOS mRNA expression in total leukocyte samples from 12 male trained subjects (TR) and a control group of 12 untrained men (UT) at rest. Relative iNOS mRNA levels (iNOS/beta-actin) were higher in UT (0.88, 0.73-1.03) when compared with TR (0.34, 0.09-0.58, P<0.001). iNOS mRNA was not detectable in 5 of the 12 TR subjects. These initial results show that the basal expression of iNOS mRNA is downregulated by moderate endurance training. Further research should clarify whether regular training also affects the responsiveness of leukocyte iNOS gene expression to stimulatory signals. It would be of interest to establish whether moderate training can exert a suppressive and therefore therapeutic effect on the elevated levels of expression of iNOS observed in, for example, several inflammatory disorders.

Transcriptional and translational regulation of heat shock proteins in leukocytes of endurance runners.

Heat shock proteins (HSP) represent cell-protective and antioxidant systems that may be induced by reactive oxygen species, cytokines, and hyperthermia. In the present study, we evaluated the influence of heavy endurance exercise and training on HSP27 and HSP70 in peripheral leukocytes of 12 athletes (before and at 0, 3, and 24 h after a half-marathon) and 12 untrained controls on protein and mRNA levels by flow cytometry and RT/PCR, respectively. HSP transcripts increased significantly immediately after acute exertion accompanied by elevated levels of corresponding proteins. HSP protein expression remained high until 24 h postexercise. Significant increases of plasma interleukin-8, myeloperoxidase, and creatine kinase occurred after exercise. Basal HSP expression was usually lower in trained compared with untrained subjects. Applying in vitro heat shock to resting blood samples of all subjects significantly stimulated HSP mRNA, showing higher increases in trained individuals. The exercise-induced alterations indicate that immunocompetent cells became activated. In addition to heat stress, other exercise-associated stress agents (oxidants, cytokines) may have also participated in stimulation of HSP expression in leukocytes. The expression pattern of HSP due to training status may be attributed to adaptive mechanisms.

Expression of the inducible nitric oxide synthase (iNOS) in human leukocytes: responses to running exercise.

INTRODUCTION: We examined the influence of two different bouts of vigorous running exercise on the expression of the inducible nitric oxide synthase (iNOS) in leukocytes (LE). METHODS: In study 1, 10 trained runners competed in a half marathon (HM) lasting 90.5 +/- 11.0 min. In study 2, 8 untrained subjects performed a graded treadmill test followed by a continuous run (CR) until exhaustion (11.3 +/- 1.3 min). iNOS mRNA levels were assessed by RT/PCR at rest, 0, 3, and 24 h after HM and CR. In study 2, iNOS was additionally analyzed at the protein level in lympho- (L), mono- (M), and granulocytes (G) by flow cytometry at rest and up to 48 h after CR. RESULTS: Analysis revealed a rise of the iNOS transcript directly after the HM in 8 of 10 subjects. In study 2, the expression of iNOS protein at rest differed between L (mean +/- SE: 30.9 +/- 4.5% iNOS positive cells), M (91.3 +/- 4.0%), and G (64.9 +/- 10.3%): 3 h after CR, expression of iNOS increased in L (67.3 +/- 7.4%) and G (90.3 +/- 2.9%) and was still elevated 48 h post-exercise. However, our measurements failed to detect significant changes of leukocyte iNOS mRNA in response to CR. After the HM, our findings were paralleled by elevated plasma levels of interleukin-8, myeloperoxidase (MPO), and partly of TNF-alpha, whereas CR only induced a low rise of MPO. CONCLUSION: Our investigations revealed an increased expression of iNOS at the transcriptional and translational level in response to vigorous exercise. This reflects an inflammatory response and may contribute to an exercise-induced rise of endogenous nitric oxide production. It remains unclear if these effects serve an in-vivo immunoregulatory or cell-damaging role.

IFN-alpha regulates IL 10 production by CML cells in vitro.

High levels of spontaneous in vitro IL 10 secretion by a subset of untreated chronic phase CML patients' cells are shown to be decreased in the presence of IFN-alpha. However, the lower level of spontaneous IL 10 secretion by healthy control cells are was not depressed by IFN-alpha. In contrast to its effects on IL 10 production, IFN-alpha increased the low spontaneous secretion of IL 1alpha by patients' cells, bud did not further increase the higher levels of spontaneous IL 1beta secretion by normal cells. It had no effect on secretion of TNF-alpha by patients or normals. Spontaneous secretion of IL-1alpha (or IFN-gamma) by patients' cells was not observed whether or not IFN-alpha was present. Therefore, one mechanism of action of IFN-alpha in vivo may involve decreasing endogenous IL 10 secretion (thereby reducing suppressive effects on T cell reactivity) and increasing IL 1beta secretion (thereby enhancing antigen presentation).

Cytokine modulation of TH1/TH2 phenotype differentiation in directly alloresponsive CD4+ human T cells.

Organ graft rejection is caused by the recognition of allogeneic MHC molecules by recipient T cells by two different pathways. The indirect pathway of alloreactivity requires the presentation of MHC peptides from the graft by autologous APC, as with conventional antigen. The direct pathway, on the other hand, requires the recognition of foreign MHC on foreign cells. The regulatory mechanisms for this component of alloreactivity have not been extensively studied. We show here that the T cell response activated by alloantigens in the direct pathway is similarly constrained and modulated by cytokines, as has been shown for classic antigen presentation. Thus, the inclusion of IL-2 or TGF-beta in MLC performed with purified responder T cells resulted in outgrowth of cells secreting IL-2 and IFN-gamma, whereas addition of IL-4, IL-10, or anti-TGF-beta encouraged outgrowth of cells secreting IL-4 and IL-10. T cells alloactivated via the direct pathway and then cloned in IL-2 alone secreted IL-4 and IL-10 as well as IFN-gamma and IL-2 (Th0 phenotype). Established clones remained susceptible to cytokine modulation, such that IL-4 and IL-10 decreased their secretion of IL-2 and IFN-gamma, whereas TGF-beta suppressed IL-4 and IL-10 secretion. The first alterations of Th0 toward Th1 or Th2 phenotypes could already be observed after only a very brief exposure to cytokines of 48 hr, followed by extended culture with IL-2 alone. These results confirm that human T cells with Th1 and Th2 phenotypes, recognizing alloantigen via the direct pathway, derive from the same IL-2-secreting precursor and can be manipulated by cytokines in an analogous fashion to conventional antigen-reactive cells. These findings may have implications for manipulating the direct pathway of alloantigen recognition in human organ transplantation.

Cellular immune responses to autologous chronic myelogenous leukaemia cells in vitro.

Using a modification of the autologous mixed lymphocyte/tumour cell culture (MLTC), it is demonstrated here that lymphocytes from chronic-phase myelogenous leukaemia (CML) patients (n = 58), but not from their HLA-identical siblings, proliferated upon coculture with autologous tumour cells. However, in most cases, the level of proliferation measured was low (stimulation index < 3, n = 37). This was most likely related to the amount of interleukin-10 (IL-10) released into the culture medium by the CML cells, because addition of neutralizing anti_IL-10 serum to MLTC markedly enhanced proliferative responses. In addition, supplementation of media with IL-1 alpha further enhanced proliferative responses and a combination of anti-IL-10 serum and IL-1 alpha was more effective than either agent alone. Only HLA-DR-matched CML cells, but not HLA-DR-mismatched CML cells or matched or mismatched PBMC restimulated proliferation of IL-2-dependent T cell lines derived from MTLC supplemented with IL-1 alpha and anti-IL-10 serum. The responding cells under these conditions were predominantly CD4+ and secreted IL-2, and interferon gamma; some secreted IL-4, but none secreted IL-10. These data therefore suggest the existence of an HLA-DR-restricted DTH/Th1-type of tumour-specific immunity in CML patients, which may be down-regulated in vitro by excessive secretion of IL-10 together with depressed secretion of IL-1.

Interleukin 10 is a human T cell growth factor in vitro.

Although interleukin (IL)-10 inhibited lymphocyte proliferation in mixed lymphocyte cultures (MLC) and blocked stimulation of alloreactive T cell clones (TCC) by peripheral blood mononuclear cells (PBMC), the cells surviving culture with IL-10 showed enhanced viability. A minority of IL-2-dependent T cell lines, moreover, incorporated tritiated thymidine when cultured with IL-10 alone; their proliferation with IL-10 was dose-dependent, prevented by addition of neutralizing antisera to IL-10 but not to IL-2 and/or IL-4 and observed both shortly (4 days) and later (7-10 days) after T cell allostimulation. Examination of the proliferative responses to IL-10 of a panel of TCC revealed heterogeneity of responsiveness: whereas only one of five CD8+ TCR2 (T cell receptor alpha, beta)-TCC proliferated with IL-10, three of five CD4+ TCR2-TCC proliferated, one of them strongly. In contrast, all three TCR1(gamma delta)-TCC tested responded to IL-10, albeit rather weakly. These results therefore suggest that in addition to its well-established inhibitory action on T cell activation, IL-10 may also exert positive influences on clonal expansion of subsets of preactivated T cells.

Human gamma delta T cells are resistant to induction of anergy but not to induction of cell death in vitro.

A condition of hyporesponsiveness can be induced in certain mature human alpha beta (TCR2) cells relatively easily by their stimulation in the absence of costimulatory signals (signal 1 alone). This state of "anergy" has been implicated in tolerance to self and transplanted organs as well as tumors and may represent an important regulatory component of immune responsiveness. Little is known about whether the same condition applies to gamma delta (TCR1) cells. We therefore undertook to investigate anergy induction in TCR1 cell clones using several approaches known to induce this state in TCR2 cells. First, TCR1 clones were found not to be anergized by culture on immobilized CD3 monoclonal antibody (mAb), while the majority of TCR2 clones were anergized. Second, blocking of autocrine proliferation (stimulated in TCR1 or TCR2 clones by mitogen in the presence of accessory cells) using CTLA-4-lg, a soluble B7 family counterreceptor resulted in anergy induction in TCR2 cells but not TCR1 cells, although experiments with CHO cells transfected with B7-1 (CD80) genes confirmed that these TCR1 clones were responsive to costimulation with B7. Third, blocking mitogen-induced proliferation with anti-IL 2 receptor antibodies and anti-IL 2 antisera resulted in anergy induction in TCR2 but not TCR1 cells. Fourth, stimulation with the calcium ionophore ionomycin also anergized TCR2 but not TCR1 cells. In all four systems, but especially in the latter, stimulation by signal 1 alone resulted in high levels of cell death (> 50%) which was similar for both TCR1 and TCR2 cells. Therefore, these data may reflect a high level of resistance to tolerance induction (manifested as proliferative anergy) but not to clonal deletion (manifested as stimulation-dependent cell death) on the part of TCR1 cells.

Extrathymic development and function of human T-lymphocytes from bone marrow cells in vitro.

To enrich low-density human bone marrow (BM) cells for putative progenitors of T-lymphocytes, CD7+ CD3- cells were sorted (purity was estimated at > 99.9%) and cultured under limiting dilution conditions with irradiated allogeneic stimulator cells, interleukin (IL) 2, and PHA. Clonal populations were available for analysis from Day 25 onward. By this time, all clones (n = 54) expressed CD3 and alpha/beta-T cell receptor (TCR2). Fifty percent of the clones were CD4+ and 50% were CD8+, with no double positives, whereas almost all clones obtained under identical conditions from peripheral blood (PB) cells were CD4+. All clones were capable of autocrine proliferation, which was blocked by CD25 or CD71 mAb. Most or all clones tested (n = 15) responded to IL 4 and IL 7 as well as IL 2, but not to IL 3 or GM-CSF and only two responded to IL 9. Most clones accumulated mRNA for GM-CSF, IL 2, IL 3, IL 4, IL 5 and also IL 9, but 6 of 11 were negative for IFN-gamma mRNA, and all were negative for IL 6 mRNA. Sixty-two percent of CD4+ and 85% of CD8+ clones (total 70% of all clones) mediated lectin-dependent cell lysis; but whereas 35% of CD4+ and 65% of CD8+ clones (total 46% of all clones) lysed K562 natural killer (NK)-susceptible targets, only 24% of CD4+ and 5% of CD8+ clones (total 17% of all clones) killed lymphokine-activated killer (LAK)-susceptible Daudi cells. Only three clones lysed allogeneic LCL targets and none lysed autologous targets. Furthermore, none of the clones proliferated when stimulated by autologous cells, neither did they suppress proliferative responses of autologous cells. These results suggest that CD3- cells from the bone marrow can acquire functional cytotoxic and proliferative programs extrathymically during in vitro culture with IL 2, mitogens and allogeneic cells, but do not manifest autoreactivity in the three test systems, cytotoxicity, suppression, or autocrine proliferation.

Induction of hyporesponsiveness in human lymphocytes-T despite their expression of both the co-receptor CD28 and its ligand B7.

A majority (42/62) of TCR2+ interleukin 2-dependent human T lymphocyte clones was found concordantly to express not only the CD28 co-receptor structure at the cell surface but also its ligand B7. Interactions between CD28 and B7 can have important consequences for T cell activation, particularly in providing "signal 2" to prevent the induction of anergy caused by stimulation via the antigen receptor only ("signal 1"). However, despite the expression of co-receptor and ligand on the same cell surface, it remained possible to induce hyporesponsiveness in T cell clones either when using CD3 antibodies to deliver signal 1 or when using other T cell clones as stimulators. Thus, the potential for intraclonal autostimulation via CD28/B7 is apparently insufficient to prevent downregulation of responsiveness in these two systems.

Evolution of a CD3+CD4+ alpha/beta T-cell receptor+ mature T-cell clone from CD3-CD7+ sorted human bone marrow cells.

In order to study extrathymic differentiation in vitro, CD7+CD3- lymphocytes were sorted from normal human bone marrow and cultured under conditions of limiting dilution together with irradiated pooled allogeneic peripheral blood mononuclear cells (PBMC) and phytohemagglutinin (PHA) in the presence of 1000 U/ml of interleukin-2 (IL-2). One clone was obtained that failed to react with monoclonal antibody (mAb) TCR delta 1 (TCR1, gamma/delta-specific) or WT31 (TCR2, alpha/beta-specific). From day 35 through day 74 in culture, the surface phenotype of this clone evolved into CD3+, CD4+, CD8-, TCR2+, TCR1-, and was further characterized as CD2+, CD45RO+, CD16-, and CD56-. The presence of mRNA for TCR alpha and beta but not gamma and delta chains was confirmed by Northern blotting. Accessory cell-dependent autocrine proliferative responses to PHA (most likely driven by IL-2) were initially absent, but became measurable at the same time as the TCR was acquired. However, in the absence of PHA, the clone failed to respond to a panel of homozygous B-cell lines representing the majority of MHC class II alleles. Autoreactivity was also not demonstrable. Cytotoxicity was limited to MHC unrestricted "natural killer (NK)-like" lysis of K562 target cells, with no autocytotoxicity detected. The NK-like lysis diminished over time in parallel with the acquisition of surface TCR. The cloned cells were not suppressive for mature lymphocyte proliferation. After stimulation, the cells secreted tumor necrosis factor alpha and granulocyte/macrophage colony-stimulating factor (GM-CSF) detected by immunoassays, and T-cell growth factors, most likely IL-2, as detected by bioassays. Polymerase chain-reaction methods demonstrated the presence of mRNA for IL-2, IL-3, IL-4, IL-9, interferon-gamma, and GM-CSF in these cells after stimulation with PHA and B-LCL. These results suggest that cells with the phenotype and some functional characteristics of mature T lymphocytes can evolve extrathymically in vitro from T-cell precursors sorted from normal human bone marrow.