TCR usage and cytokine expression in peripheral blood and BAL T cells

T cells are thought to play an important regulatory role in atopic asthma. We hypothesized that human blood and BAL T cell subsets bearing various TCR-Vbeta genes might show selective differences in their cytokine profile. Peripheral blood (PB) and bronchoalveolar lavage (BAL) T cells from seven atopic asthmatic and six non-atopic non-asthmatic subjects were stimulated with PMA and ionomycin in the presence of monensin and analysed for TCR-Vbeta expression and production of cytokines at the single cell level. The percentage of IFN-gamma- and IL-2-producing BAL T cells was elevated compared with PB T cells from both the asthmatic subjects and the non-atopic, non-asthmatic controls. A small percentage of PB and BAL T cells produced IL-4 and IL-5, in asthmatic and normal subjects. In peripheral blood, the percentage of T cells expressing each cytokine was similar in the various TCR-Vbeta subsets and in total CD3+ T cells in all normal and six of seven asthmatic subjects. However, there was a substantial degree of heterogeneity in the cytokine profile of BAL TCR-Vbeta subsets compared with the total CD3+ T cells. This was more obvious in the asthmatic subjects with a reduction in the percentage of IFN-gamma- and IL-2-expressing T cells (five of seven asthmatic subjects) and an increase in the percentage of IL-4- and IL-5-expressing T cells (two of seven asthmatic subjects). These data confirm previous findings of an elevated proportion of IFN-gamma- and IL-2-producing BAL T cells while only a small proportion of PB and BAL T cells produce IL-4 and IL-5. Moreover, subsets of BAL T cells, defined by their TCR-Vbeta usage, may differ in their cytokine profile compared with the total CD3+ T cells, implying that T cells expressing different Vbeta elements may play different roles in regulating the airway inflammation in asthma.     

Reduced number and function of peripheral dendritic cells in coeliac disease

Dendritic cells (DC) play a pivotal role in shaping the immune response in both physiological and pathological conditions. In peripheral blood at least two subsets, the myeloid and plasmacytoid, have been described as having different T stimulatory functions and a variable degree of maturation. Certainly, antigen presentation plays a crucial role in the pathogenesis of coeliac disease and circulating immune cells are thought to reflect the state of immune response within the gut. Therefore, we aimed to investigate the quantitative and phenotypical modifications of peripheral blood DC, together with their functional properties, in this pathological condition. Blood samples from 11 untreated patients before and after a course of gluten-free diet, 27 treated patients and 14 controls underwent flow-cytometric analysis, while immunomagnetically sorted DC from the CD patients and eight human leucocyte antigen (HLA)-DQ2/8(+) bone marrow donors were used to evaluate maturation status through the CD83 expression, cytokine profile for interleukin (IL)-6, IL-10, IL-12 and interferon (IFN)-alpha by enzyme-linked immunosorbent assay (ELISA), and functional properties by mixed leucocyte reaction before and after pulsing with digested gliadin. We found that in both untreated and treated patients, a significant reduction of the entire DC population, mainly the plasmacytoid subset, in comparison to healthy controls was observed. In active disease, an impaired allogenic lymphocyte reaction and a significant reduction of IFN-alpha production, paralleled by the presence of a more immature status, were also demonstrated. All the latter modifications have been reverted by pulsing DC with digested gliadin.     

Whole virus influenza vaccine activates dendritic cells (DC) and stimulates cytokine production by peripheral blood mononuclear cells (PBMC) while subunit vaccines support T cell proliferation

Three types of trivalent influenza vaccines were analysed for their in vitro stimulatory properties on immune cells from young healthy volunteers. A whole inactivated virus (WV) vaccine, a conventional subunit (c-SU) preparation and a new virosomal subunit (v-SU) vaccine were used. Blood-derived DC up-regulated MHC class II, CD54, CD80 and CD86 after exposure to WV vaccine, indicating their functional maturation, but were only moderately affected by subunit (SU) vaccines. In addition, IL-12 and tumour necrosis factor-alpha (TNF-α) secretion by DC were markedly enhanced by WV, but not by SU vaccines. The production of IL-2 and interferon-gamma (IFN-γ) by PBMC was also strongly stimulated by WV, but much less by SU vaccines, among which the v-SU vaccine was a better stimulator of IL-2 secretion. In contrast to WV vaccine both SU vaccines were powerful stimulators of PBMC proliferation. Our results suggest that the presence of influenza core components leads to the activation of DC and triggers the production of cytokines by PBMC. SU vaccines are in contrast excellent stimulators of T cell growth. A combination of WV and SU vaccines in immunization regimes might allow optimal T cell priming as well as the efficient generation and maintenance of memory cells.     

Cord blood mesenchymal stem cells propel human dendritic cells to an intermediate maturation state and boost interleukin‐12 production by mature dendritic cells

Pathogen‐derived entities force the tissue‐resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord blood an immature MSC population was identified. Remarkably, these immature stem cells modulated DCs in a different way. Marker expression was unchanged during the differentiation of monocytes towards immature DCs (iDCs) when cocultured with cord blood MSC [unrestricted somatic stem cells (USSCs)]. The maturation to mature DCs (mDCs) was enhanced when DCs were co‐cultured with USSC, as evidenced by the up‐regulation of costimulatory molecules. Endocytosis of dextran by iDCs was hampered in the presence of USSCs, which is indicative for the maturation of iDCs. Despite this maturation, the migration of iDCs cocultured with USSCs appeared to be identical to iDCs cultured alone. However, USSCs increased the migration of mDCs towards CCL21 and boosted interleukin‐12 production. So, USSCs mature iDCs, thereby redirecting the antigen‐uptake phenotype towards a mature phenotype. Furthermore, DC maturation by lipopolysaccharide (LPS) or USSCs reflects two distinct pathways because migration was unaffected when iDCs were matured by coculture with USSCs, while it was strongly enhanced in the presence of LPS. DCs are able to discriminate the different MSC subtypes, resulting in diverse differentiation programmes.     

Increased intracellular T helper 1 proinflammatory cytokine production in peripheral blood, bronchoalveolar lavage and intraepithelial T cells of COPD subjects

The role of T cells in the pathophysiology of chronic obstructive pulmonary disease (COPD) is not yet certain, although varying reports have shown increases in T helper 1 (Th1) and/or Th2 cytokines in peripheral blood and bronchoalveolar lavage (BAL). No studies have examined cytokine production by intraepithelial T cells obtained by bronchial brushing (BB). Intracellular cytokine analysis of T cell subsets from peripheral blood, BAL and BB from smoker and ex-smoker COPD patients, COPD patients receiving inhaled corticosteroids and smoker and non-smoker control subjects was studied using multi-parameter flow cytometry. CD4 : CD8 inversion was noted in the peripheral blood of smoker and ex-smoker COPD groups, in BAL and BB from smoker controls and BAL of COPD smokers. There was an increase in intracellular CD8(+) T cell Th1 proinflammatory cytokines in some COPD groups in the peripheral blood and in CD8(+) T cell tumour necrosis factor (TNF)-alpha in some COPD groups and smoker controls in BAL and BB. There was an increase in proinflammatory cytokines in COPD smokers compared with ex-smokers and a decrease in COPD smokers receiving inhaled corticosteroids in the airways. There was a negative correlation between forced expiratory volume in 1 s (FEV(1)) and the percentage of BAL and intraepithelial CD8(+) T cells producing TNF-alpha. COPD patients exhibit systemic inflammation as evidenced by increased intracellular Th1 proinflammatory cytokines in blood, BAL and intraepithelial CD8(+) T cells, whereas smoker controls showed localized Th1 response in the lung only. Systemic therapeutic targeting of TNF-alpha production by CD8(+) T cells may improve morbidity in COPD patients while targeting of TNF-alpha in the lung may prevent smokers progressing to COPD.     

非小细胞肺癌患者外周血树突状细胞的数量变化分析

检测不同分期非小细胞肺癌患者外周血树突状细胞(PBDC)的数量差异及术后PBDC的数量变化,并探讨其临床意义。用流式细胞仪检测10名健康人、44例非小细胞肺癌患者PBDC亚群DC1和DC2。与正常健康人群相比,非小细胞肺癌患者外周血DC1/DC2的数量明显减少,并且分期越晚PBDC的数量越少;与术前基线相比,术后1个月时PBDC的数量均出现了一定程度的恢复,进一步检测发现Ia期患者在术后6月时PBDC数量接近正常水平。因此可以认为非小细胞肺癌细胞可导致PBDC数量减少,DC治疗前对肿瘤患者适当地减轻肿瘤负荷有可能提高抗肿瘤疗效。     

Testosterone inhibits immunoglobulin production by human peripheral blood mononuclear cells

We studied the in vitro effect of testosterone on spontaneous immunoglobulin production by human peripheral blood mononuclear cells (PBMC). Testosterone inhibited IgG and IgM production by PBMC both from males and females. The inhibitory effect of testosterone was revealed at doses more than 1 nm, increased dose-dependently, and reached a plateau at 100 nm. At doses <1000 nm, testosterone did not reduce cell viability. Testosterone treatment reduced IgG production by 59.0% and that of IgM by 61.3% compared with control. Immunoglobulin production by B cells was also suppressed by testosterone, though the magnitude of the suppressive effect on B cells was lower than that on whole PBMC; testosterone-induced decrease of IgG production compared with control was 26.9% and that of IgM was 24.9%. Exogenous IL-6 partially restored the impaired immunoglobulin production of testosterone-treated PBMC; IgG production in testosterone culture was increased by IL-6 from 35.6% to 66.5% of control and that of IgM was also increased from 38.9% to 71.2%, respectively. Testosterone treatment reduced IL-6 production of monocytes by 78.4% compared with control, but neither affected that of T cells or B cells. These results suggest that testosterone may suppress immunoglobulin production of human PBMC directly by inhibiting B cell activity and indirectly by reducing IL-6 production of monocytes. It is thus indicated that this hormone may have protective and therapeutic effects on human autoimmune diseases.     

高血压病患者外周血树突状细胞亚群的变化

目的研究高血压病患者外周血树突状细胞（DCs）的表达情况及其在高血压病发生发展过程中的作用。方法用流式细胞仪四色荧光标记技术检测29例高血压病（HT）患者（实验组）及31例非高血压患者（对照组）外周血树突状细胞亚群的比例及数量,评价组间差异,并分析高血压病患者外周血浆细胞样树突状细胞（pDC）与血压（blood pressure,BP）的关系。结果高血压病（HT）患者与血压正常患者相比,外周血中髓样树突状细胞（mDC）比例[（7.917±4.296）‰比（8.18±5.669）‰]及绝对数[（6.971±2.115）×107/L比（7.123±5.387）×107/L]均降低,但差异无统计学意义（P〉0.05）。而浆细胞样树突状细胞（pDC）比例[（1.239±0.669）‰比（1.897±0.859）‰]及绝对数[（1.794±2.244）×107/L比（2.819±4.997）×107/L]明显升高,且差异有统计学意义（P〈0.05）,高血压病患者的外周pDC数量与收缩压（SBP）、舒张压（DBP）均成呈正相关关系（r=0.424及0.487,P〈0.05）,pDC比例与SBP、DBP无明显相关（P〉0.05）。结论高血压病的发生发展中存在着炎症反应和免疫活化,树突状细胞可能在该过程中发挥一定的作用。     

Differing effects of clarithromycin and azithromycin on cytokine production by murine dendritic cells

Summary The macrolide antibiotics are now well known to have anti-inflammatory effects. Because dendritic cells (DCs) orchestrate immune responses, we examined the in vitro effects of clarithromycin (CAM), azithromycin (AZM) and midecamycin (MDM) on the expression of co-stimulatory molecules and production of cytokines [interleukin (IL)-10, IL-6, interferon (IFN)-gamma, IL-12p40, tumour necrosis factor (TNF)-alpha] of murine bone marrow-derived DCs by lipopolysaccharide (LPS) stimulation. A 15-membered macrolide, AZM, and a 14-membered macrolide, CAM, significantly enhanced the intensity of a co-stimulatory molecule, CD80, on DCs but not CD86 and CD40. AZM significantly increased the production of IL-10 and CAM significantly inhibited the production of IL-6 by DCs. However, a 16-membered macrolide, MDM, did not have any significant effect on these surface markers and cytokine productions. Moreover, AZM increased IL-10 and CAM decreased IL-2 productions significantly, when naive T cells derived from spleen were co-cultured with DCs treated in advance with LPS and these macrolides. These findings suggest that 14-membered and 15-membered, but not 16-membered macrolides play as anti-inflammatory agents, at least in part, through modulating the functions of DCs. However, each macrolide affects them in different ways.     

The role of thymus in the aging of Th cell subpopulations and age-associated alteration of cytokine production by these cells

Mouse CD4⁺ T cells were subdivided into two subpopulations,naive (CD44^low CD45RB^high) and memory (CD44^highCD45RB^low) Tcells, by flow cytometric analysis. Examination of spleen andperipheral blood of C57BL/6 mice of various ages revealed thatthere was a reciprocal ageassociated change in these two subpopulations,i.e. naive T cells predominant in young mice decreased withage, while memory T cells increased. In order to investigatethe role of the thymus in the age change of naive and memoryT cells, we employed two experimental systems: radiation bonemarrow chimeras constructed between young and old mice, andgrafting of young or old thymus into nude mice. Data from thesetwo experiments suggested that the young thymus has a greaterability to provide naive T cells than the old thymus, whilethe old thymus favors the maintenance of memory T cells ratherthan naive T cells. In reference to cytokine production by enrichednaive and memory T cells, young naive T cells produced mainlyIL-2 and young memory T cells mainly IL-4. On the other hand,in old mice, memory T cells produced twice as much IL-2 thannaive T cells, although the level was significantly lower thanthat of young mice. In addition, old naive T cells producedtwice as much IL-4 than old memory T cells. These results suggesteda distinct age change in the profile of cytokine productionand functional heterogeneity of two T_h cell subpopulations.     

T cell proliferation, MHC class II restriction and cytokine products of gliadin-stimulated peripheral blood mononuclear cells (PBMC).

The immune response of PBMC to gliadin was investigated in patients with coeliac disease (CoD) by examining proliferation, MHC restriction and cytokine production. Gliadin induced low levels of proliferation in 63% of eight untreated patients, 32% of 28 treated patients and 35% of 31 healthy control subjects. In MHC restriction studies, the proliferative response to gliadin was inhibited (range 47-98% inhibition) in the presence of a MoAb to HLA-DR in each of three coeliac and three control donors studied. Using flow cytometry, increased expression of activation markers (HLA-DR and IL-2R) was demonstrated on gliadin-stimulated T cells from four of nine coeliac patients and three of seven healthy control donors. Cytokines were studied in culture supernatants using ELISA. Gliadin was a potent inducer of IL-6 and IL-10 in 100% of coeliac patients and controls, whereas IL-4 was not produced in either subject group. Gliadin induced IL-2 production in 40% of untreated patients, 42% of treated patients and 35% of healthy control donors. Interferon-gamma (IFN-gamma) in gliadin-stimulated cultures was found only in coeliac patients, observed in 33% of untreated patients and 25% of treated patients. Spontaneous secretion of both IL-2 and IFN-gamma was found more frequently in patients with untreated disease (87% of cases versus 21% of controls for IFN-gamma and 40% versus 0% for IL-2). These results suggest, as manifest by IFN-gamma production, that gliadin stimulates a Th1/Th0-like response in coeliac patients and a Th0-like response in healthy controls.     

Bioenergetic analysis of human peripheral blood mononuclear cells

Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)−1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses.     

Distribution of MHC class II antigens in feline tissues and peripheral blood

A new monoclonal antibody raised against gradient-purified feline immunodeficiency virus was found to recognize a bimolecular complex, comprising 27-29 kD and 32-35 kD subunits, on feline peripheral blood lymphocytes. Immunoperoxidase staining of feline tissues with this antibody, designated 43.2H2, demonstrated a reactivity pattern similar to that described for MHC II antigens of the dog, horse, and pig, but differed from human and mouse in having staining of T-cell zones in spleen and lymph nodes. Flow cytometric analysis revealed that 42.3H2 reacted with 88.97% +/- 16.00% of feline peripheral blood lymphocytes (n = 20). This high level of reactivity was found to be consistent by repeated sampling over a 4-month period. Two-color flow cytometric analysis was used to determined the reactivity pattern on lymphocyte subsets: 88.92% +/- 7.30% of CD4+ lymphocytes were 42.3H2-positive, while 85.99% +/- 11.46% of CD8+ cells were positive (n = 11 for both). B lymphocytes had the highest reactivity (99.47% +/- 0.45; n = 9) and also had the highest fluorescence intensity. By gating based on light scatter properties, 95.06% +/- 7.35% of monocytes were 42.3H2-reactive (n = 18), while granulocytes were negative.     

Intracellular cytokine production in peripheral blood lymphocytes: a comparison of values in infertile and fertile women

Citation
Horká P, Jaro?ová R, Malí?ková K, Janatková I, Mare?ková H, Zima T, Kalousová M. Intracellular cytokine production in peripheral blood lymphocytes: a comparison of values in infertile and fertile women. Am J Reprod Immunol 2011; 65: 466–469 Problem To analyze the relation of the fertility and pregnancy of women of childbearing age to the intracellular (IC) production of tumor necrosis factor alpha (TNF‐α), interferon gamma (IFN‐γ), and interleukins 2 and 4 (IL‐2 and IL‐4). Method of study Intracellular cytokine production in peripheral blood CD3⁺ CD4⁺ lymphocytes was analyzed by flow cytometry in 185 women being treated for infertility and 50 fertile women of childbearing age. Results Infertile women have a significantly higher IC production of TNF‐α, IFN‐γ, IL‐2, and IL‐4 and higher ratios of TNF‐α/IL‐2, TNF‐α/IL‐4, and TNF‐α/IFN‐γ compared to the fertile women. Conclusion Cytokines produced by Th lymphocytes are important in orchestrating the immune response during conception, and Th‐cell dysregulation could be a reason for infertility.     

A distinct pattern of cytokine production from blood mononuclear cells in multitransfused patients with β-thalassaemia

The unstimulated and induced production of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), IL-3, IL-6, stem cell factor (SCF), IL-1β, tumour necrosis factor-alpha (TNF-α), TNF-β, interferon-gamma (IFN-γ) and transforming growth factor-beta (TGF-β) was determined after culture of blood mononuclear cells from 22 patients with severe β-thalassaemia in a regular transfusion programme, five non-regularly transfused patients with β-thalassaemia intermedia and nine normal persons. A distinct pattern of cytokine production in thalassaemic patients was detected, namely a low unstimulated production of all cytokines and a significant increase in the stimulated production of IFN-γ, TNF-α and IL-1β; these abnormalities were more pronounced in the more heavily transfused older patients. The increased production of the above cytokines, which usually characterize the acute response to infectious agents and have a negative effect on erythropoiesis, may explain the deterioration of anaemia found in thalassaemic patients during acute infections.     

T-cell subset analysis of cryopreserved human peripheral blood mononuclear cells

A criticism of current techniques for monitoring changes in T-cell subset numbers over extended periods in individuals with disease states in which such changes might provide insight is the fact that serial samples taken are usually analysed fresh and therefore not in the same assay. To try to overcome this problem we have stored peripheral blood mononuclear cells frozen in liquid nitrogen and thence examined their ability to form sheep red blood cell (E) rosettes and to label with OK monoclonal antibodies. Results obtained show that cell viabilities following freezing and T-cell subset analysis of E-rosette positive cells are no different when fresh or frozen and subsequently thawed peripheral blood mononuclear cells are used.     

Incomplete activation of peripheral blood dendritic cells during healthy human pregnancy

Successful pregnancy relies on the adaptation of immune responses that allow the fetus to grow and develop in the uterus despite being recognized by maternal immune cells. Dendritic cells (DCs) are central to the control of immune tolerance, and their state of activation at the maternal-decidual interface is critical to the feto-maternal immunological equilibrium. So far, the involvement of circulating DCs has been investigated poorly. Therefore, in this study we investigated whether, during healthy human pregnancy, peripheral blood DCs (PBDCs) undergo changes that may be relevant to the adaptation of maternal immune responses that allow fetal tolerance. In a cross-sectional study, we analysed PBDCs by six-colour flow cytometry on whole blood samples from 47 women during healthy pregnancy progression and 24 non-pregnant controls. We demonstrated that both myeloid and plasmacytoid PBDCs undergo a state of incomplete activation, more evident in the third trimester, characterized by increased expression of co-stimulatory molecules and cytokine production but lacking human leucocyte antigen (HLA)-DR up-regulation. To investigate the contribution of soluble circulating factors to this phenomenon, we also performed culture experiments showing that sera from pregnant women added to control DCs conditioned a similar incomplete activation that was associated with reduced DC allostimulatory capacity, supporting the in vivo relevance of our findings. We also obtained evidence that the glycoprotein hormone activin-A may contribute to DC incomplete activation. We suggest that the changes of PBDCs occurring during late pregnancy may aid the comprehension of the immune mechanisms operated by the maternal immune system to maintain fetal tolerance.     

T helper 1 (Th1)/Th2 cytokine expression shift of peripheral blood CD4+ and CD8+ T cells in patients at the post-acute phase of stroke

Local humoral and cellular immune responses modulate the inflammatory processes involved in the development of atherosclerotic lesions, as well as in the evolution of brain infarcts in stroke patients. The role of systemic adaptive immunity on the progression of such disease manifestations is less clear. In the current study, we evaluated the percentages of T helper 1 (Th1) [interleukin (IL)-2, interferon (IFN)-gamma] and Th2 (IL-4, IL-10) cytokine-producing peripheral blood CD4+ and CD8+ T cells in 23 patients with a history of ischaemic stroke (IS) at the chronic stable phase of the disease (median post-stroke time 34.5 months). Seven stroke-free individuals matched for age and vascular risk factors (matched controls, MC) were collected for comparison. To measure cytokine values at baseline and after stimulation, we used a flow cytometry method of intracellular cytokine staining. Intrinsic Th1 and Th2 cytokine production in unstimulated T cells was negligible in all study participants. Following mitogenic stimulation with phorbol 12-myristate13-acetate/ionomycin, both the IS and the MC groups exhibited a similarly strong Th1 response; IL-2 production predominated in the CD4+ T cells and IFN-gamma in the CD8+ T cells. However, when measuring the Th2 cytokine-production capacity post-stimulation, a significant increase in the percentage of IL-4-producing T cells was observed in the IS groups, compared with the MC group, resulting in a significantly lower ratio of IFN-gamma-/IL-4-producing T cells. No such Th2 enhancement could be confirmed for the case of IL-10. We propose that in IS patients there is a systemic shift of the immune system towards Th2 responses at the late post-acute phase of stroke.     

Haemorrhage-induced alterations in function and cytokine production of T cells and T cell subpopulations.

Haemorrhage produces alterations in macrophage, T and B cell function. In order to better define the mechanism for the effects of blood loss on immune response, we examined function of and cytokine production by purified T cells, CD4+ and CD8+ subpopulations after blood loss. Whereas T and CD4+ cells from control, unhaemorrhaged animals produced no alteration in proliferation when added to cultures of mitogen-stimulated splenocytes from normal mice, proliferation was decreased when T or CD4+ cells from haemorrhaged mice were included. The addition of CD8+ cells from haemorrhaged animals to mitogen-stimulated cultures reduced proliferation by approximately 50% more than that found when CD8+ cells from control, unhaemorrhaged animals were included. Supernatants of mitogen-stimulated splenocytes from haemorrhaged mice contained significantly less IL-2 and interferon-gamma (IFN-gamma) than did those from control, unhaemorrhaged mice. CD4+ populations from haemorrhaged mice produced significantly more IL-10, and significantly less IFN-gamma, than did CD4+ cells from control, unhaemorrhaged mice. There were no significant differences in IL-2, IL-4, IL-10 or IFN-gamma production by CD8+ cells from haemorrhaged or control mice. The present experiments demonstrate that haemorrhage affects both CD4+ and CD8+ T cell subsets. In particular, haemorrhage appeared to activate CD4+, Th2 cells, with concomitant suppression of the Th1 subpopulation. These results provide a mechanism which may contribute to the alterations in cytokine production previously described to occur following blood loss.     

Cell cycle traverse in AHH-1 tk +/− human lymphoblastoid cells exposed to the chromosomal mutagen,m-amsa

AHH-l tk +/− cells were exposed to the chemotherapeutic agent, m-amsa, both in complete medium and in medium without serum, subcultured in complete medium, and the effect on the traverse of the cell cycle determined by flow cytometric analysis of bromodeoxyuridine (BrdUrd)-labeled DNA. After exposure to m-amsa (day 0), the percentage of S-phase cells increased significantly (P < 0.0017) with increasing concentration. Cells also accumulated in G2/M as evidenced by the significant (P < 0.0026), concentration-dependent increase in the percentage of cells detected within this phase. Serum deprivation during exposure resulted in significantly (P = 0.024) more cells in S-phase than in cultures exposed to m-amsa in complete medium. After three days in culture, a significant (P = 0.0001) accumulation of cells in G2/M was present; the percentage of cells in G2/M did not differ significantly (P = 0.148) in cultures exposed to m-amsa in complete medium or in serum-free medium. However, a significant (P < 0.001) loss of S-phase cells was found in cultures exposed without serum. At day 7, no significant concentration effects were detected (G0/G1, P = 0.6026; S-phase, P = 0.9773; G2/M, P = 0.8401). These results demonstrate that exposure to m-amsa perturbs the traverse of the cell cycle, initially by inhibiting the completion of S-phase and followed by an accumulation of cells in G2/M. In addition, exposure to m-amsa under conditions of serum deprivation results in an increased percentage of cells in the initial S-phase after exposure, the loss of S-phase cells from the culture after three days, and the appearance of a subdiploid peak, consistent with cells undergoing apoptosis. © 1996 Wiley-Liss, Inc.