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.     

In Vitro Inhibition of Cellular Immune Responses by Benzodiazepines and Pk 11195 Effects on Mitogen-and Alloantigen-driven Lymphocyte Proliferation and on IL-1, IL-2 Synthesis and IL-2 Receptor Expression

Abstract

In vitro mitogen-driven lymphocyte proliferation tests (Con A, LPS) on murine lymph node and spleen cells revealed inhibition of T and B cell stimulation by different benzodiazepines and by PK 11195, with IC50 values in the low micromolar range. T cell responses as a consequence of recognition of alloantigens, as measured in mixed lymphocyte cultures (MLC), were affected in an analogous way. In all systems, agonists at peripheral type benzodiazepine receptors (Ro 5–4864 and the non-benzo-diazepine compound PK 11195) and diazepam which acts on both, central and peripheral type benzodiazepine receptors, were most potent; clonazepam, a central type agonist, proved about half as active. the central type antagonist Ro 15–1788 failed to antagonize the action of diazepam and clonazepam. Variations among cells from several congenic strains of mice were modest. Cytotoxicity could not be made responsible for drug effects. the most susceptible stage of mitogen-triggered T and B lymphocyte proliferation was found to be at incipience. Radioresistant, adherent spleen cells, upon LPS-stimulation formed only small amounts of the cytokine IL-1. Its release was affected only at very high drug concentrations. Similar small amounts of IL-1 were generated during MLC; in this case, the drugs were about 10times less potent than in mitogen-induced proliferation assays. Peripheral agonists were more active on IL-1 synthesis. Spleen cells stimulated with Con A and cultivated with the highest concentration of diazepam and clonazepam formed markedly greater amounts of IL-2 than those cultivated in medium, while at this concentration PK 11195 allowed no formation of the lymphokine. Flow cytometry revealed increased formation of IL-2R for 10-⁴M diazepam, Ro 5–4864 and PK 11195, with little difference to medium controls for other compounds and molarities. IL-2 formation during MLC was inhibited by central and peripheral agonists with IC50s in the micro-molar range. In presence of diazepam, these MLC cells displayed frequencies of IL-2R similar to those stimulated with Con A.     

In Vitro Inhibition of Cellular Immune Responses by Benzodiazepines and Pk 11195 Effects on Mitogen-and Alloantigen-driven Lymphocyte Proliferation and on IL-1, IL-2 Synthesis and IL-2 Receptor Expression

In vitro mitogen-driven lymphocyte proliferation tests (Con A, LPS) on murine lymph node and spleen cells revealed inhibition of T and B cell stimulation by different benzodiazepines and by PK 11195, with IC50 values in the low micromolar range. T cell responses as a consequence of recognition of alloantigens, as measured in mixed lymphocyte cultures (MLC), were affected in an analogous way. In all systems, agonists at peripheral type benzodiazepine receptors (Ro 5-4864 and the non-benzo-diazepine compound PK 11195) and diazepam which acts on both, central and peripheral type benzodiazepine receptors, were most potent; clonazepam, a central type agonist, proved about half as active. the central type antagonist Ro 15-1788 failed to antagonize the action of diazepam and clonazepam. Variations among cells from several congenic strains of mice were modest. Cytotoxicity could not be made responsible for drug effects. the most susceptible stage of mitogen-triggered T and B lymphocyte proliferation was found to be at incipience. Radioresistant, adherent spleen cells, upon LPS-stimulation formed only small amounts of the cytokine IL-1. Its release was affected only at very high drug concentrations. Similar small amounts of IL-1 were generated during MLC; in this case, the drugs were about 10times less potent than in mitogen-induced proliferation assays. Peripheral agonists were more active on IL-1 synthesis. Spleen cells stimulated with Con A and cultivated with the highest concentration of diazepam and clonazepam formed markedly greater amounts of IL-2 than those cultivated in medium, while at this concentration PK 11195 allowed no formation of the lymphokine. Flow cytometry revealed increased formation of IL-2R for 10-⁴M diazepam, Ro 5-4864 and PK 11195, with little difference to medium controls for other compounds and molarities. IL-2 formation during MLC was inhibited by central and peripheral agonists with IC50s in the micro-molar range. In presence of diazepam, these MLC cells displayed frequencies of IL-2R similar to those stimulated with Con A.     

Lipoprotein-Dependent and -Independent Immune Responses to Spirochetal Infection

In this study, we used the epidermal suction blister technique, in conjunction with multiparameter flow cytometry, to analyze the cellular and cytokine responses elicited by intradermal injection of human volunteers with synthetic analogs for spirochetal lipoproteins and compared the responses to findings previously reported from patients with erythema migrans (EM). Compared with peripheral blood (PB), lipopeptides derived from the N termini of the Borrelia burgdorferi outer surface protein C and the 17-kDa lipoprotein of Treponema pallidum (OspC-L and 17-L, respectively) elicited infiltrates enriched in monocytes/macrophages and dendritic cells (DCs) but also containing substantial percentages of neutrophils and T cells. Monocytoid (CD11c⁺) and plasmacytoid (CD11c⁻) DCs were selectively recruited to the skin in ratios similar to those in PB, but only the former expressed the activation/maturation surface markers CD80, CD83, and DC-SIGN. Monocytes/macrophages and monocytoid DCs, but not plasmacytoid DCs, displayed significant increases in surface expression of Toll-like receptor 1 (TLR1), TLR2, and TLR4. Staining for CD45RO and CD27 revealed that lipopeptides preferentially recruited antigen-experienced T-cell subsets; despite their lack of antigenicity, these agonists induced marked T-cell activation, as evidenced by surface expression of CD69, CD25, and CD71. Lipopeptides also induced significant increases in interleukin 12 (IL-12), IL-10, gamma interferon, and most notably IL-6 without corresponding increases in serum levels of these cytokines. Although lipopeptides and EM lesional infiltrates shared many similarities, differences were noted in a number of immunologic parameters. These studies have provided in situ evidence for a prominent “lipoprotein effect” during human infection while at the same time helping to pinpoint aspects of the cutaneous response that are uniquely driven by spirochetal pathogens.     

Lymphocyte Responses to Chemokines

Today, almost three dozen human chemokines have been identified. The main function of these soluble proteins is the recruitment of leukocytes to sites of infection and inflammation. This review emphasizes the new developments in the field of lymphocyte responses to chemokines. Notably, it was shown that lymphocytes require stimulation to become responsive to chemokines, a process that is closely linked to chemokine receptor expression. As an exception, one chemokine, SDF-1, is a highly effective chemoattractant for non-activated T lymphocytes and progenitor B cells. Of particular interest are the chemokines IP10 and Mlg which bind to a receptor with selective expression in activated T lymphocytes and, therefore, may be critical mediators of T lymphocyte migration in T cell-dependent immune-responses. All other chemokines with activities in lymphocytes do also induce responses in monocytes and granulocytes. The involvement of chemokine receptors in HIV infection is briefly mentioned, while other interesting areas in chemokine research, such as hematopoiesis and angiogenesis, are not discussed.     

IL-5 Promoter Polymorphism Enhances IgE Responses to Staphylococcal Superantigens in Adult Asthmatics

Interleukin 5 (IL-5) is a key cytokine involved in the induction of T-helper type 2 (Th2) responses in the asthmatic airway. We investigated IL-5 genetic polymorphisms associated with asthma phenotypes, including IgE responses to staphylococcal enterotoxins A and B (SEA and SEB, respectively), in asthmatics. Adult asthmatics (n=310) and normal controls (n=160) were enrolled in the present study. Serum total and specific IgE to SEA and SEB were measured. Two IL-5 polymorphisms, -746A>G and +4499T>G, were genotyped using the primer-extension method. There were no significant differences in genotype or haplotype frequencies of these polymorphisms between the two groups. Asthmatics carrying the AG/GG genotype at -746A>G had a significantly higher prevalence of serum specific IgE to SEA (P=0.008), higher total IgE levels (P=0.014), and lower PC20 methacholine levels (P=0.002) compared to those with the AA genotype. These findings suggest that the IL-5 promoter polymorphism at -746A>G enhances serum total and specific IgE responses to SEA, which may augment airway hyperresponsiveness in adult asthmatics.     

Modulation of Murine Lymphocyte Mitogen Responses by Glycerol-Teichoic Acid

Glycerol-teichoic acid (GTA) showed a modulatory effect on the in vitro response of murine splenocytes to the mitogens concanavalin A (Con A) and lipopolysaccharide (LPS) as measured by incorporation of ³H-thymidine. GTA inhibited the response to Con A when added prior to addition of the mitogen, while addition 24 hr after had no significant effect on the response. The degree of suppression was dose dependent in a range from 0.1-5μg GTA/culture. The spleen cell response to LPS was enhanced by GTA when added prior to the mitogen. Peak enhancement occurred at 1-2 μgGTA/culture, depending on the time of addition. GTA added 24 hr after LPS produced no significant effect on mitogenesis. Addition of GTA alone to spleen cell cultures produced a slight suppression of DNA synthesis and was toxic at 10 μg/culture if incubated at least 66 hr. GTA is bound to murine spleen cells as indicated by decreased passive hemagglutination inhibition activity of culture supernates.     

Modulation of Murine Lymphocyte Mitogen Responses by Glycerol-Teichoic Acid

Glycerol-teichoic acid (GTA) showed a modulatory effect on the in vitro response of murine splenocytes to the mitogens concanavalin A (Con A) and lipopolysaccharide (LPS) as measured by incorporation of ³H-thymidine. GTA inhibited the response to Con A when added prior to addition of the mitogen, while addition 24 hr after had no significant effect on the response. The degree of suppression was dose dependent in a range from 0.1-5μg GTA/culture. The spleen cell response to LPS was enhanced by GTA when added prior to the mitogen. Peak enhancement occurred at 1-2 μgGTA/culture, depending on the time of addition. GTA added 24 hr after LPS produced no significant effect on mitogenesis. Addition of GTA alone to spleen cell cultures produced a slight suppression of DNA synthesis and was toxic at 10 μg/culture if incubated at least 66 hr. GTA is bound to murine spleen cells as indicated by decreased passive hemagglutination inhibition activity of culture supernates.     

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.     

Histamine Blocks Interleukin 2 (IL-2) Gene Expression and Regulates IL-2 Receptor Expression

Histamine inhibited the roliferative response of human peripheral blood mononuclear cells (PBMCT to the T cell mitoFen Phytohema P (PHA-P) in a dose-dependent fashion. This inhibition was mePutiated via the H₂ receptor since cimetidine, a known H₂ antagonist, removed the inhibition, whereas the addition of the H₁ antagonist Diphenhdramine did not. Inhibition occurred durin the inductive phase of the cell cycle, since histamine added 24 hours aBter PHA-P stimulation had no effect on subsequent T cell proliferation, and was attributable to inhibition of interleukin-2 (IL-2) gene expression. Both secreted IL-2 and messenger RNA coding for IL-2 were inhibited by histamine. In contrast, histamne exerted no inhibitory effect on the expression of cell surface receptors for IL-2 as determined by flow cytometry. Furthermore, histamine-treated cells retained full responsiveness to exogenously administered IL-2, which completely reversed the anti-proliferative effect of histamine. In some donors, histamine enhanced the ercentage of IL-2 receptor positive cells. Stimulated PBMC from AIDS KS patients as a group, displayed a lower ercentage of IL-2 receptor bearing cells, which was significantly increased gy the addition of histamine even at concentrations as low as 10^--⁶ M and peaking at 10^--³ M. These findings indicate that histamine exerts its anti-proliferative effects on T cells by inhibiting IL-2 production, via blockade of IL-2 gene expression. In addition, histamine seems to exert immunomodulating effects on IL-2 receptor expression, particularly in those individuals with AIDS-KS.     

Lymphocyte Proliferation Responses Induced to Broadly Reactive Th Peptides Did Not Protect against Equine Infectious Anemia Virus Challenge

The effect of immunization with five lipopeptides, three containing T-helper (Th) epitopes and two with both Th and cytotoxic T-lymphocyte (CTL) epitopes, on equine infectious anemia virus (EIAV) challenge was evaluated. Peripheral blood mononuclear cells from EIAV lipopeptide-immunized horses had significant proliferative responses to Th peptides compared with those preimmunization, and the responses were attributed to significant responses to peptides Gag from positions 221 to 245 (Gag 221-245), Gag 250-269, and Pol 326-347; however, there were no consistent CTL responses. The significant proliferative responses in the EIAV lipopeptide-immunized horses allowed testing of the hypothesis that Th responses to immunization would enhance Th and CTL responses following EIAV challenge and lessen the viral load and the severity of clinical disease. The EIAV lipopeptide-immunized group did have a significant increase in proliferative responses to Th peptides 1 week after virus challenge, whereas the control group did not. Two weeks after challenge, a significant CTL response to virus-infected cell targets occurred in the EIAV lipopeptide-immunized group compared to that in the control group. These Th and CTL responses did not significantly alter either the number of viral RNA copies/ml or disease severity. Thus, lipopeptide-induced proliferative responses and enhanced Th and CTL responses early after virus challenge were unable to control challenge virus load and clinical disease.     

Post Recognition ION Dependent Events in Mitogen Induced Lymphocyte Proliferation and in Cytotoxic Effector Cell Responses

This review discusses the increased permeability and enhanced uptake transport of monovalent and divalent cations following mitogen-induced lymphocyte roliferation. The observed Ca2⁺ uptake is discussed in terms of gated Ca2⁺ channels. The importance of divalent cations, particularly Ca2⁺, is discussed in terms of triggering of cytotoxic effector cell responses for three model systems (antibody dependent cytotoxicity, mitogeninduced cytotoxicity and cell mediated cytotoxicity); (85).     

Post Recognition ION Dependent Events in Mitogen Induced Lymphocyte Proliferation and in Cytotoxic Effector Cell Responses

This review discusses the increased permeability and enhanced uptake transport of monovalent and divalent cations following mitogen-induced lymphocyte roliferation. The observed Ca2⁺ uptake is discussed in terms of gated Ca2⁺ channels. The importance of divalent cations, particularly Ca2⁺, is discussed in terms of triggering of cytotoxic effector cell responses for three model systems (antibody dependent cytotoxicity, mitogeninduced cytotoxicity and cell mediated cytotoxicity); (85).     

Polyclonal Lymphocyte Responses to Murine Trypanosoma cruzi Infection

Intraperitoneal infection of young adult C57BL/6 males with 10(5) blood or cloned culture forms of Trypanosoma cruzi (CL strain) induced the appearance in spleen, blood, and lymph nodes of cytotoxic effector cells detectable in a lectin-dependent 51Cr-release assay. The effector cells were conventional cytotoxic T lymphocytes (CTL), since they were Thy 1+ and Lyt 2+, and the lysis of tumour target cells was strictly dependent on the presence of lectin. CTL activity is already detectable in spleen 2 days after infection, reaches a peak at 2 weeks, and returns to normal levels during the chronic phase (1 month onwards). Increased levels of CTL activity were also detected in lymph nodes with similar kinetics, even in animals that were splenectomized prior to infection. In contrast to spleen, significant levels of CTL activity persisted in lymph nodes in the chronic phases. This functional variable correlates with the appearance of high numbers of large Lyt 2+ lymphocytes in the same organs (50 to 100-fold higher than in control, uninfected mice). Very similar responses are detected in a T. cruzi sensitive mouse strain (C3H/HeJ). It appears, therefore, that T. cruzi infection results in a large polyclonal activation of Lyt 2+ lymphocytes, some of which differentiate to effector, cytolytic functions.     

Polyclonal Lymphocyte Responses to Murine Trypanosoma cruzi Infection

Lymphoid activity was studied in spleen and lymph node cells from Trypanosoma cruzi-infected mice. Blast transformation in each lymphocyte class was assessed by dual parameter analysis for size and surface markers by both FACS and conventional immunofluorescence, while proliferative activity was measured by tritiated thymidine uptake, autoradiography, and analysis of DNA content in single cells. Acute infection results in rapid blast transformation and proliferative activity of all three lymphocyte classes (Ig+, L3T4+, and Lyt 2+). At 2 weeks of infection most cells in these organs are enlarged and more than half are dividing. By 2 and 6 months after infection (chronic phase of resistant strains), large numbers of activated B lymphocytes and, to a lesser extent, of Lyt 2+ T cells are still detected. Similar results were obtained in C57BL/6 (resistant) and C3H/HeJ (susceptible) mouse strains. The implications of this massive polyclonal lymphocyte response to the parasite for the physiopathology of acute and chronic infection are discussed.     

Shipment Impairs Lymphocyte Proliferative Responses to Microbial Antigens

Lymphocyte proliferation assays (LPAs) are widely used to assess T-lymphocyte function of patients with human immunodeficiency virus infection and other primary and secondary immunodeficiency disorders. Since these assays require expertise not readily available at all clinical sites, specimens may be shipped to central labs for testing. We conducted a large multicenter study to evaluate the effects of shipping on assay performance and found significant loss of LPA activity. This may lead to erroneous results for individual subjects and introduce bias into multicenter trials.     

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.     

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.     

Lipoteichoic Acid Inhibits Interleukin-2 (IL-2) Function by Direct Binding to IL-2

Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved.