Glutamine, Lymphocyte Proliferation and Cytokine Production

The present in vitro study was conducted to examine how glutamine influences the lymphocyte function. Glutamine had no effect on the production of interleukin-1β, interleukin-6 or tumour necrosis factor-α, but influenced the production of interleukin-2 and interferon-γ. Glutamate, leucine, isoleucine and valine (substrates for glutamine production), or the combination of glutamate and leucine, did not influence the lymphocyte proliferative response or the cytokine production. In conclusion, glutamine influenced the production of some T-cell-derived cytokines, and is thereby important for optimal lymphocyte proliferation. Furthermore, the results show that lymphocytes are not capable of producing glutamine.     

Immunologic Specificity of Lymphocyte Cell Lines from Dogs Exposed to Beryllium Oxide

Abstract

We have reported that dogs exposed twice to aerosols of beryllium oxide (BeO) developed Be-specific immune responses within the lung, along with granulomatous and fibrotic lung lesions. To evaluate the specificity of the immune response, lymphocytes from lungs and blood of BeO-exposed dogs were co-cultured over an irradiated blood monocyte layer, alternately with interleukin 2 and BeSO₄. Resultant cell lines were then tested for their response to different metal cations, common canine recall antigens, and BeSO₄ in an in vitro cell proliferation assay. The cell lines responded to BeSO₄ in a dose-dependent fashion, with mean stimulation indices of 7, 58, 119, and 112 at concentrations of 0.01, 1.0, 10, and 100 μM BeSO₄ respectively. Cells not proliferate when incubated with ZnSO₄ or NiSO₄, or with canine distemper, leptospira, adenovirus 2, parvovirus, or parainfluenza antigens. Lymphocytes from normal vaccinated dogs proliferated markedly when cultured with these antigens. Cells from the cultured cell lines (91%) stained with Thy-1 (a pan T-cell marker) and 96% stained with DT2 (a helper T-cell marker). Furthermore, the Be-induced proliferative response was restricted by major histocompatibility (MHC) class II antigens. These data reinforce the premise that inhalation exposure of dogs to BeO produces lung lesions and MHC class II restricted immunologic responses mediated by Be-specific, helper T-Cells. These data further confirm the hypothesis that antigen localized to the lung results in the recruitment of T-cells to the lung, followed by localized antigen-specific, cell-mediated immune responses.     

Spontaneous Lymphocyte Proliferation during Trauma and Infection

Spontaneous lymphocyte proliferation (SpP), measured in vitro as the rate of [14C]thymidine incorporation in blood lymphocytes, was investigated in non-infected postoperative patients, infected postoperative patients, and healthy volunteers, with 72, 24, and 3 h of lymphocyte culture. With 24-h cultures, infected postoperative patients showed 17-fold higher SpP than non-infected postoperative patients (2527 +/- 1552 versus 151 +/- 77 cpm, mean +/- SD, P less than 0.001) and 37-fold higher SpP than healthy volunteers (P less than 0.001). Postoperative patients without infection had twice as high SpP as healthy volunteers (P less than 0.001). Lymphocytes harvested after 24 h of cell culture showed significantly higher SpP than corresponding values at 72 and 3 h, in patients as well as in healthy volunteers (P less than 0.01). Infected postoperative patients showed a higher SpP than non-infected patients after only 3 h of cell culture (270 +/- 192 versus 48 +/- 10 cpm, P less than 0.001). An inverse correlation was observed between the level of SpP and body temperature in patients with postoperative infection (r = -0.62, P less than 0.05). The results indicate that lymphocytes are activated by uncomplicated surgery and particularly by postoperative infection, and that characteristics of SpP are reproducible in short cell-culture periods, which suggests that in vitro measurements of SpP may be of value in the detection of severe postoperative infection.     

CTLA4Ig对丝裂原诱导的淋巴细胞反应的影响

本研究证明CTLA4Ig 与B7 分子结合后, 可以阻断B7 与CD28 的结合。CTLA4Ig 可以抑制丝裂原所触发淋巴细胞的增殖反应, 同时抑制细胞因子IL 2 ,IL 4 的产生以及IL 2R 的表达。表明CTLA4Ig 可以通过不同的途径影响T淋巴细胞的活化     

Effects of Dynorphin on the Pha-Induced Lymphocyte Proliferation in Vitro

The effect of the opioid peptide dynorphin (DYN) on PHA-induced lymphocyte proliferation has been evaluated in the present study.

A significant increase in PHA-induced lymphocyte activation was observed when DYN was added to cultures 48 hr after the mitogenic stimulation. This effect occurred at suboptimal (3.12 and 6.25 ug/ml) PHA concentrations and at DYN doses ranging from 10^-9 to 10^-12 M. Conversely, DYN did not affect the lymphocyte blastogenic process either when added before, or simultaneously or after intense PHA (12.5 ug/ml) stimulation.

Naloxone preincubation did not modify the above described effects.

Results suggest that DYN might play a role in neuroendocrine regulation of the lymphocyte blastogenic process.     

Pseudomonas aeruginosa Exoenzyme S Stimulates Murine Lymphocyte Proliferation In Vitro

The exuberant immunoinflammatory response that is associated with Pseudomonas aeruginosa infection is the major source of the morbidity and mortality in cystic fibrosis (CF) patients. Previous studies have established that an exoproduct of P. aeruginosa (exoenzyme S) is a mitogen for human T lymphocytes and activates a larger percentage of T cells than most superantigens, which may contribute to the immunoinflammatory response. An animal model would facilitate studies of the pathophysiologic consequences of this activation. As a first step toward developing an animal model, the murine lymphocyte response to exoenzyme S was examined. When stimulated with exoenzyme S, splenocytes isolated from naive mice entered S phase and proliferated. The optimum response occurred after 2 to 3 days in culture, at 4 x 10(5) cells per well and 5.0 micrograms of exoenzyme S per ml. The response was not due to lipopolysaccharide, since Rhodobacter sphaeroides lipid A antagonist did not block the response. Other preparations of exoenzyme S stimulated lymphocyte proliferation, since the response to recombinant exoenzyme S (rHisExo S) cloned from strain 388 was similar to the response to exoenzyme S from strain DG1. There was evidence that genetic variability influenced the response, since A/J, CBA/J, and C57BL/6 mice were high responders and BALB/cJ mice were low responders following stimulation with exoenzyme S. Both splenic T and B lymphocytes entered the cell cycle in response to exoenzyme S. Thus, murine lymphocytes, like human lymphocytes, respond to P. aeruginosa exoenzyme S, which supports the development of a murine model that may facilitate our understanding of the role that exoenzyme S plays in the pathogenesis of P. aeruginosa infections in CF patients.     

Nitric Oxide Modulates Lymphocyte Proliferation But Not Secretion of IL-2

The objective of this study was to determine the effects of nitric oxide (NO) on lymphocyte proliferation and cytokine release. Bronchoalveolar lavage (BAL) cells served as the source of NO and were obtained from rats treated with a single, intratracheal dose of bleomycin (3.6 mg/kg). At the time of sacrifice, the spleens were removed and the lymphocytes separated. Co-cultures containing BAL cells, lymphocytes and concanavalin-A were established and incubated at 37°C for 24 hours at which time proliferation, nitrite concentration and interleukin-2 (IL-2) production were measured. At ratios from 5:1 to 1:4 (BAL:lymphocyte) there was a significant reduction in lymphocyte proliferation. There was a significant, negative correlation between NO concentration and thymidine incorporation which was reversed when the NO synthase inhibitor N^G-monomethyl-L-arginine (NMA) was added to the co-cultures. Despite marked inhibition of spleen lymphocyte proliferation by NO₂ released by BAL cells, there was no corresponding reduction in IL-2 production. These data demonstrate that macrophages, activated in vivo, produce NO which regulates lymphocyte growth but not necessarily functions such as the secretion of the cytokine IL-2. Further, the ability of IL-2-dependent CTLL-2 cells to proliferate in the presence of excess IL-2 was also inhibited by BAL cells, confirming that NO inhibits lymphocyte growth.     

Catalase and Lipopolysaccharide Enhance Proliferation in the Rat Mixed Lymphocyte Reaction

Proliferation of rat spleen cells in a mixed lymphocyte culture was amplified fivefold or more in the presence of 2000 units of catalase/ml, as measured by [3H]thymidine incorporation. A similar effect was observed with 1 microgram of lipopolysaccharide (LPS)/ml. Addition of polymyxin B abrogated the promotional effect of LPS, but not that of catalase. These results indicate that the hydrogen peroxide generated by some cells in the rat spleen cell mixed lymphocyte culture suppresses the proliferative response. The demonstration that removal of plastic adherent cells (reducing the percentage of monocytes/macrophages by 75-80%) also results in a 5- to 10-fold increase in a subsequent MLR, indicates that some of the adherent cells may be the producers of hydrogen peroxide, which at higher concentrations suppresses the T-cell proliferation. The enhanced proliferation was not mainly due to increased interleukin 2 (IL-2) production, since the IL-2 concentrations of catalase and LPS-containing cultures were lower than those of control cultures.     

In Vitro Phencyclidine-Induced Inhibition of Lymphocyte Proliferation: Prevention by Cell Activation

The proliferative response of phytohemagglutinin (PHA)- and interleukin 2 (IL-2)-activated murine splenocytes was studied in the presence of phencyclidine (PCP), a potent psychotomimetic drug of abuse. PCP inhibited [3H]-thymidine incorporation in lymphocytes treated with PHA or IL-2. This inhibitory action was dependent upon the drug doses and the time of incubation with the cultures. There was no significant inhibitory activity of PCP when it was added 24 hrs or 48 hrs after mitogenic stimuli. Parallely, a lower inhibitory effect was observed when IL-2 or PHA were simultanously present in the incubation medium. Moreover, the pretreatment for 18 hrs with IL-2 completly counteracted PCP-induced depression of PHA-stimulated lymphocytes. We suggest that PCP affects some pathway that regulates the activation of resting T cells rather than affecting already cycling cells.     

The Effects of Cocaine and Stress on Lymphocyte Proliferation in Rats1

The effects of cocaine, stress, and the combination of cocaine and stress on proliferative responses to mitogens in vitro were examined in rats. In this paradigm, hypothalamic dopamine and norepinephrine were measured to examine whether catecholamine levels relate to changes in immune function. Cocaine, stress, and cocaine plus stress decreased cellular immune function to Con A mitogen compared with a control group. Hypothalamic dopamine levels were inversely related to immune proliferation to lipopolysaccharide (LPS) mitogen. The results are discussed in terms of the deleterious effects of cocaine and stress and possible mechanisms for cocaine and stress induced immune suppression.     

Blood Lymphocyte Proliferation Response to Pollen Extract as a Monitor of Immunotherapy

In a study of immunotherapy 41 children with seasonal rhinoconjunctivitis due to deciduous tree pollen allergy were monitored by means of symptom scoring, patient self-evaluation, conjunctival provocation tests and lymphocyte proliferation in vitro to the allergen. The lymphocyte responsiveness to birch pollen decreased significantly during the first year of immunotherapy. However, neither the lymphocyte responsiveness before treatment nor changes in lymphocyte reactivity during the immunotherapy correlated with the clinical efficacy of the therapy as evaluated by changes in symptom scores, self-evaluation or conjunctival provocation test changes in the individual patients. The results indicate the lymphocyte responsiveness to an allergen cannot be used to select patients for immunotherapy, i.e. to predict whether a patient would benefit from immunotherapy or not, or to evaluate the effects of immunotherapy after beginning the treatment. However, lymphocyte proliferation response to an allergen indicates clinical sensitivity.     

Comparison of Human Immunodeficiency Virus Antigens as Stimulants for Lymphocyte Proliferation Assays

CD4 proliferative responses to the human immunodeficiency virus (HIV) type 1 (HIV-1) p24 (gag) antigen inversely correlate with the plasma viral load in HIV-infected subjects who control viral replication without antiretroviral therapy. Use of a single HIV-1 protein to assess CD4 proliferative responses may not reflect the global response to this pathogen. We compared the abilities of HIV p24 and gp120 antigens from two different vendors, an inactivated whole HIV-1 MN virion preparation and an HIV-1E culture supernatant antigen, to elicit proliferative responses in HIV-seropositive and HIV-seronegative donors. Peripheral blood mononuclear cells from 12 HIV-seropositive donors (each with HIV-1 loads <4,000 copies/ml of plasma, >350 CD4 T lymphocytes/mm³, and no antiretroviral therapy) and 15 HIV-seronegative donors were assessed with multiple concentrations of each stimulant by standard lymphocyte proliferation assays. Wide variations in response rates were found, with zero, three, five, and eight individuals demonstrating stimulation indices of >3 for the HIV culture antigen supernatant, gp120, p24, and inactivated whole-virus preparations, respectively. These results suggest that the use of the inactivated whole virus resulted in a more sensitive assay for detection of CD4 T-lymphocyte function in HIV-infected subjects.     

Human Lymphocyte Proliferation Responses following Primary Immunization with Rabies Vaccine as Neoantigen

Evaluation of the T-cell immune response following primary antigenic challenge with a neoantigen is a critical aspect of assessment of the cellular immune response. While many antigens can be used to accurately assess in vitro T-cell proliferation to a recall antigen, only a few neoantigens have been tested for their capacities to measure T-cell responses in vitro to a primary immunization. Rabies vaccination is an excellent candidate for the testing of T-cell proliferation responses to a primary immunization because few individuals have been exposed to rabies virus antigens. In the present study 14 rabies vaccine-naïve, healthy adult volunteers were immunized against rabies virus, and T-cell proliferation and antibody responses were measured before and after vaccination. Optimal lymphocyte proliferation to soluble rabies virus antigen occurred after 8 days in culture. The average level of uptake of tritiated thymidine postimmunization was 29,620 ± 4,448 cpm, whereas preimmunization levels were 12,660 ± 3,448 cpm (P = 0.002). All individuals showed increases in rabies virus antibody titers from <0.05 to 5.59 ± 1.64 IU/ml. The degree of proliferation to tetanus toxoid as a recall antigen was similar to the response to rabies virus antigen among the cohort. Due to high levels of preimmunization proliferation, four subjects failed to demonstrate a twofold increase in response to rabies virus antigen. The high levels of T-cell responses may be due to a viral superantigen effect in some individuals. Rabies vaccination offers a safe and effective means for measurement of both T- and B-cell immune responses to a neoantigen in healthy and immune suppressed individuals.     

Hydatidiform Mole Macromolecules Inhibit Interleukin-2-Mediated Murine Lymphocyte Proliferation In Vitro

ABSTRACT: Macromolecules extracted from hydatidiform mole trophoblast inhibit mitogen-induced lymphocyte proliferation. To characterize the mechanism of this immunomodulation, we determined the effects of hydatidiform mole vesicle fluid (HMF) and tissue extracts (HME) on lymphokine function in vitro. Utilization of interleukin-1 (IL-1) and interleukin-2 (IL-2) were determined by using a lymphoma cell line (LBRM-33-1A5) and a murine T cell line (CTLL2), respectively. HMF suppressed (P < .05) IL-2-dependent CTLL2 cell proliferation at 500 (36.4% of controls) and 50 (74.9% of controls) μg/ml. HME also suppressed CTLL2 proliferation (P < .05) at 500 (46.0% of controls), 100 (67.2% of controls), 50 (71.5% of controls), and 10 (85.4% of controls) μg/culture ml. In contrast, HMF exhibited no effect on IL-1-stimulated LBRM-33-1A5 production of IL-2. However, 500 μg/ml of HME inhibited (P < .05) IL-2 production (63.0% of controls) in the IL-1 utilization assay. This suppressive effect was probably due to a carry over of HME from the LBRM-33-1A5 culture to the target cells (CTLL2) used to measure IL-2 production. Molecular weight chromatography of an HME sample eluted an IL-2 inhibitor in a low molecular weight (35–50 kd) and high molecular weight (> 250 kd) fraction. These data suggest that one way in which macromolecules derived from hydatidiform mole could interfere with in vitro immunologic responses is by modulating interleukin-2 function.     

人胚胸腺激素抑制组分对成人淋巴细胞增殖的影响

在研究人胚胸腺素的基础上,通过对提取工艺的改革,获得了人胚胸腺素抑制组分及促进组分。我们用~3H—TdR掺入DNA法观察了人胚胸腺素抑制组分对成人淋巴细胞增殖的影响,试验结果表明,呈现明显的剂量依赖关系,即40μg的人胚胸腺素抑制组分对PHA诱导的淋巴细胞增殖有显著的抑制作用(P<0.001),当稀释至2μg或更低浓度时,淋巴细胞的增殖恢复到正常范围。而正常淋转无论是高浓度或稀释到较低浓度,都未显示出明显的抑制或促进作用。     

Effect of Anesthesia on a Whole Blood Lymphocyte Proliferation Assay in the Rat

Lymphocyte proliferation assays are commonly used to quantify the effects of immunosuppressive drugs in animal models, but the influence of anesthetic agents on those assays is not well understood. We used a whole blood proliferation assay to compare lymphocyte proliferation in blood drawn from normal male Lewis rats that were sedated using three common methods. Rats (n = 12) were serially bled from the orbital plexus while anesthetized with diethyl ether, methoxyflurane, or carbon dioxide. Before the beginning of the anesthetic trials, a random subset of the rats (n = 6) was bled via the jugular vein using only manual restraint to provide a baseline control group. A comparison of the lymphocyte proliferation results obtained under these four conditions (manual restraint, diethyl ether, methoxyflurane, or CO₂) showed no significant differences. The only hematological variation seen was an elevation of the number of circulating lymphocytes when ether was used. We conclude that there is no justification for withholding sedation when bleeding rats for this type of lymphocyte proliferation. Furthermore, when considering the use of one of the agents examined in this study, the method can be chosen based on factors other than potential adverse effects on the assay results.     

Synergistic Interaction Between Dehydroepiandrosterone and Prednisolone in the Inhibition of Rat Lymphocyte Proliferation

The interaction between dehydroepiandrosterone (DHEA) and prednisolone (PD) in the inhibition of rat lymphocyte proliferation was evaluated. The in vitro proliferative response of splenocytes from male Sprague-Dawley rats stimulated with phytohemagglutinin (PHA) was measured by the incorporation of ³H-thymidine into the DNA. DHEA and PD were added at the initiation of cultures individually and in various molar ratio combinations. The search for synergy additivity or antagonism between the two compounds was performed by using the median-effect method of Chou and Talalay and Drewinko's statistical analysis. Both compounds individually inhibit the proliferation of PHA-stimulated rat lymphocytes. DHEA with an IC₅₀ value of 13.4 pM was a thousand times less potent than PD which had an IC₅₀ value of 5.4 nM. Synergy was observed between DHEA and PD. The intensity of the interaction appeared to be function of the molar ratio of the two drugs. The association of DHEA and PD could produce enhanced steroid effects in anti-inflammatory therapy.     

Combined Inhibition Effects of Tacrolimus and Methylprednisolone on in Vitro Human Lymphocyte Proliferation

Tacrolimus (FK 506) has synergistic immunosuppressive effects in combination with corticosteroids. A steroid dose-lowering effect can be explained partly by the inhibition by FK 506 of cytochrome P-450 III A, which metabolizes corticosteroids. We investigated the influence of combinations of FK 506 and methylprednisolone (MPL) on the suppression of in vitro human lymphocyte proliferation. to quantify the type of drug interaction (synergistic, additive, and antagonistic), three methods (Drewinko's statistical analysis, median-effect principle, and normal distribution model) were used. the interaction appeared synergistic in most combination ratios, but the extent of synergism varied depending on the quantitative method. There may be optimal combination ratios of FK 506 and MPL which achieve more effective immunosuppressive effects.     

Production of a Suppressor of Lymphocyte Proliferation by Two Human Oral Carcinoma Cell Lines

This study examined the production of an immunosuppressive factor by the KB and H191 human oral squamous carcinoma cell lines. Conditioned media (CM) from both cell lines markedly inhibited mitogen- and alloantigen-induced proliferation of normal human and rat peripheral blood lymphocytes. By contrast, the proliferation of an exponentially-growing fibroblast cell line remained unchanged by CM. The immunosuppressive factor appeared to act after lymphocyte commitment as indicated by continued blast cell formation, the failure of CM to suppress resting lymphocytes and the fact that CM caused maximum inhibition of lymphocyte proliferation 72 h after the addition of PHA. The addition of exogenous IL-2 did not counteract lymphocyte suppression. Inclusion of indomethacin and isoniazid during cell culture did not significantly alter the degree of suppressive activity. Mycoplasma contamination was absent and CM did not act directly with the thymidine or mitogen. The factor was heat stable at 50 degrees C, acid labile and had a molecular weight in excess of 300 kDa. The results demonstrate that human oral squamous carcinoma cell lines produce an immunosuppressive factor that may have a role in tumour evasion of the host immune response.