Release Of Th2 Cytokines Research Articles

Low levels of interferon‐γ in nasal fluid accompany raised levels of T‐helper 2 cytokines in children with ongoing allergic rhinitis

The T-helper 2 (Th2) cytokines interleukin-(IL-) 4, IL-5, IL-6, IL-10 and the Th1 cytokine IFN-gamma and their associations with eosinophil, eosinophil cationic protein (ECP) and immunoglobulin (Ig) E were studied in nasal lavage fluid from 60 school children with allergic seasonal rhinitis and 36 nonatopic healthy controls, before and during the pollen season. Eosinophil differential counts and IgE increased significantly in the patients during the pollen season. The eosinophil differential counts, ECP and IgE were all significantly higher during the season than in specimens simultaneously obtained from the nonatopic controls. Before season, the levels of ECP and IgE, but not eosinophils, were significantly higher in the patients than in the controls. During the season the nasal lavage fluid levels of IFN-gamma were significantly lower and the IL-4/IFN-gamma quotients significantly higher in the allergic than in the control children. In the allergic children, but not in the controls, the nasal fluid levels of the Th2 cytokines IL-4, IL-5 and IL-10 increased during the season, and together with IL-6, were correlated with the differential counts of eosinophils, and with the levels of ECP and IgE. These findings are compatible with the hypothesis that a deficient release of the Th1 cytokine IFN-gamma plays an important role in the pathogenesis of allergic inflammation. Regardless of whether the defective IFN-gamma secretion is primary or a consequence of suppression by other cytokines, it will in the atopic subjects enhance the release of Th2 cytokines, which in turn will facilitate the development of allergic inflammation.

Effect of T-helper 1 cytokines on secretion of T-helper 2 cytokines by term trophoblast cells in culture

A successful pregnancy has been postulated to be the result of a discrete balance between T-helper 1 (Th1) and T-helper 2 (Th2) type cytokines involved in growth and development of the conceptus. The aim of the present study was to examine the effect of Th1 cytokines (interleukin- 1β (IL- 1β) and tumor necrosis factor-alpha (TNFα)) on the release of Th2 cytokines including IL-6 and IL-10 by trophoblast cells obtained from term placenta. Trophoblast cells isolated by enzymatic disaggregation and Percoll gradient fractionation were cultured in supplemented medium alone or with varying concentrations of the selected recombinant cytokines. After 48 h of incubation, samples of the culture supernatant were analyzed for the Th2 cytokines IL-6 and IL-10 using specific ELISA assays. Both IL-1β and TNFα had no effect on the cell number and viability as determined by MTT assay. IL-1β significantly stimulated trophoblast release of IL-6 in a dose-dependent manner (3.3-, 5.5-, 10.3- and 22 4-fold higher compared to the control at 10, 50, 100, 500 U IL-1β/ml respectively, p < 0.05). TNFα also stimulated release of IL-6 by these cells. However, the stimulation at lower concentrations was not very high and a significant (p < 0.05) stimulation was observed only at higher concentrations (1.1-, 1.3-, 2.6- and 5.9-fold higher at 500, 1000, 1500, 2000 U TNFα/ml respectively). In contrast, neither IL-1β or TNFα exerted any significant effect on IL-10 release by term trophoblast cells (p > 0.05). The results of this study provide evidence that production of Th2 cytokines might be under the control of different regulatory pathways.

T Cell Memory Against Colon Carcinoma Is Long-Lived in the Absence of Antigen

Eradication of established colon carcinoma metastases is a major goal for adjuvant immunotherapy of this disease. This was accomplished in a murine model by targeting IL-2 to the tumor microenvironment with a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2). The generation of a long-lived protective immunity was demonstrated by a 10- to 14-fold increase in CTL precursor (pCTL) frequency and induction of genes encoding Th1 cytokines, followed by the generation of tumor-specific CD8+ T effector cells, some of which differentiated into long-lived T memory cells. The frequency of pCTL correlated with enhanced immune protection against tumor cell challenge, and long-lived T cell memory was maintained in syngeneic SCID mice in the absence of tumor Ag. Tumor cell challenge of these SCID mice, concomitant with a boost of two noncurative doses of huKS1/4-IL-2 fusion protein, resulted in the generation of primed CD8+ T effector cells with concurrent release of Th1 cytokines. These events culminated in the complete rejection of the tumor cell challenge and prevention of pulmonary metastases. Taken together, the data suggest that T cell memory against colon carcinoma can be maintained in the absence of Ag.

Suppression of anti-CD3-induced interleukin-4 and interleukin-5 release from splenocytes of mesocestoides corti-infected BALB/c mice by phosphodiesterase 4 inhibitors

We investigated the suppressive effects of rolipram, RP 73401 (piclamilast), and other structurally diverse inhibitors of adenosine 3′5′-cyclic monophosphate (cAMP)-specific phosphodiesterase (PDE4) on anti-CD3-stimulated interleukin (IL)-4 and IL-5 generation by splenocytes from BALB/c mice infected with Mesocestoides ( M) corti. RP 73401 ( ic 40: 0.011 ± 0.004 μM) was a very potent inhibitor of anti-CD3-induced IL-4 release, being ∼40-fold more potent than (±)-rolipram ( ic 40: 0.43 ± 0.09 μM). A maximal inhibition of 60–70% of the response was achieved at the top concentrations of RP 73401 (1 μM) and rolipram (100 μM). These PDE inhibitors also suppressed IL-5 generation over the same concentration ranges, but the maximal suppression achieved was only 30–40%. R-(−)-rolipram ( ic 40: 0.39 ± 0.09 μM) was ∼6-fold more potent than S-(+)- rolipram ( ic 40: 2.6 ± 0.95 μM) in inhibiting IL-4 release. A close correlation ( r 2 = 0.82) was observed between suppression of IL-4 release by PDE inhibitors and inhibition of CTLL cell PDE4, a form against which R-(−)-rolipram displayed relatively weak inhibitory potency. A poorer correlation ( r 2 = 0.26) was observed between suppression of IL-4 release and affinities of cAMP PDE inhibitors for the high-affinity rolipram binding site in mouse brain membranes. The cGMP-inhibited PDE (PDE3) inhibitor, siguazodan, had little or no effect ( ic 40 > 100 μM) on anti-CD3-stimulated release of either IL-4 or IL-5 and did not significantly enhance the inhibitory action of RP 73401 on the release of either of these cytokines. Finally, RP 73401 ( ic 50: 0.41 ± 0.19 nM) inhibited anti-CD3-stimulated DNA synthesis in splenocyte preparations from M. corti-infected mice and siguazodan (10 μM) had no effect on this response, either alone or in combination with the PDE4 inhibitor. The results show that PDE4 inhibitors suppress the release of Th2 cytokines from anti-CD3-stimulated murine spenocytes and that this effect is correlated with inhibition of a low-affinity PDE4 form.

The emerging role of immunoregulation of fibrinogen-related procoagulant Fgl2 in the success or spontaneous abortion of early pregnancy in mice and humans.

Abortion of chromosomally normal embryos in the CBA X DBA/2 mating combination is triggered by release of Th1 cytokines (tumor necrosis factor [TNF]-alpha, interferon [IFN]-gamma, and interleukin [IL]-1), which cause abortion via a novel prothrombinase, Fgl2, and polymorphonuclear leukocytes. The site of activation may be maternal vascular endothelium on arteries and veins nourishing the placenta. Activation of coagulation is also prominent in spontaneous abortion of chromosomally normal human embryos. We asked where is Fgl2 up-regulated in the uterus in murine abortions, and if similar Fgl2 expression occurs in human pregnancy failure. Control CBA X DBA/2 pregnant mice, or from mice injected with TNF-alpha + IFN-gamma on day 7.5 of gestation, were removed on day 8.5, fixed, sectioned, and subject to in situ hybridization for Fgl2. Sections were also stained for fibrin. Elective first trimester termination samples or biopsies taken early in the course of a recurrent miscarriage were similarly fixed, sectioned, and analyzed by in situ hybridization. Control and cytokine-treated mice were anticoagulated with heparin, an activator of antithrombin III, and/or the direct anti-thrombin inhibitor hirudin. Low level Fgl2 expression localized to basal decidua remote from the embryo was noted in control mice; cytokine treatment, which causes greater than 80% of abortions, produced a striking up-regulation in this area as well as in a band at the junction of decidua and myometrium. Trophoblast also became strikingly positive. Fgl2 expression was associated with increased fibrin staining. Anticoagulation significantly protected against abortions, but doses were limited by the complication of retroplacental hemorrhage. In tissue from normal first trimester pregnancy, minimal Fgl2 positivity was seen in some villous syncytiotrophoblast, in villous stroma, cytotrophoblast, and in some cells in decidua. In spontaneous abortion of normal embryo, striking Fgl2 positivity was seen in syncytiotrophoblast and extravillous cytotrophoblast, in association with areas of thrombus formation. Fgl2 appears to be physiologically expressed and may protect against the internal danger of maternal and/or fetal bleeding during pregnancy and at parturition; a role in inhibiting transplacental traffic is also possible. External dangers in the form of stress, endotoxin, and antigens eliciting Th1 cytokine responses upregulate Fgl2 prothrombinase in trophoblast as well as in decidua, which results in spontaneous abortion of immunogenetically "weaker" embryos.

Splenic Immune Suppression in Sepsis: A Role for IL-10-Induced Changes in P38 MAPK Signaling

Background.Studies have indicated that following the induction of sepsis, there is a late (24 h) generalized suppression of the immune response which is associated with increased anti-inflammatory mediator release (e.g., IL-10). However, the mechanisms by which this occurs are unknown. In this regard, recent studies indicate that p38 mitogen-activated protein kinase (p38 MAPK) may play a central role in transducing the signals from immunosuppressive agents which in turn may alter lymphoid cytokine release. The aim of this study, therefore, was to determine whether the anti-inflammatory mediator IL-10 alters splenocyte IL-2 and IFN-γ release, as well as the expression and activation of p38 MAPK in septic animals.Materials and methods.Splenocytes (SPL) (or for some experiments purified T cells) were harvested from mice subjected 24 h earlier to either sepsis by cecal ligation and puncture (CLP) or Sham-CLP and stimulated with 2.5 μg concanavalin A (ConA)/ml in the presence or absence of either monoclonal antibody (Mab) to IL-10 (4 μg/ml) or IgG control. In subsequent studies, sepsis was induced in C57BL/6J and C57BL/6 IL-10 knockout mice, and SPL harvested and stimulated with ConA. SPL cytokine release was measured by ELISA, and the expression and phosphorylation of p38 MAPK were measured by Western analysis.Results.The results indicate that Th1 cytokine (IL-2, IFN-γ) release was depressed by sepsis, while p38 MAPK expression and activity were increased in SPL as well as in T-cells. Neutralization of IL-10 byin vitrouse of anti-IL-10 Mab and in the IL-10 knockout animal restored the Th1 response and caused a downregulation of p38 MAPK expression and activity after CLP. Thus, IL-10 appears to contribute to the increase in p38 MAPK activity and expression and the corresponding suppression of Th1 response seen in late sepsis.

Th1- and Th2-type cytokines regulate the expression and production of eotaxin and RANTES by human lung fibroblasts.

Eosinophils (Eos) and fibroblasts are known to play a major role in the pathogenesis of bronchial asthma and fibrotic lung disease. Therefore, we investigated whether Th1 and Th2 cytokines stimulate the production of Eo-activating chemokines by lung fibroblasts. Analyses of the culture supernatant using multiple steps of high-performance liquid chromatography demonstrated that interleukin (IL)-4 preferentially stimulates lung fibroblasts to secrete a peak of eosinophil chemotactic activity (ECA) which, upon N-terminal analyses, showed similar sequence to eotaxin, whereas interferon (IFN)-gamma had negligible effect on the release of this chemokine. In contrast, tumor necrosis factor (TNF)-alpha stimulated lung fibroblasts to release two peaks of activity that were found to correspond to eotaxin and regulated on activation, normal T cells expressed and secreted (RANTES), respectively. Interestingly, IL-4 synergized with TNF-alpha to increase greatly the production of three biochemically distinct eotaxin forms. In contrast, IFN-gamma synergized with TNF-alpha to increase RANTES production. Neither IL-2, IL-5, IL-6 nor IL-10 had an effect on lung fibroblasts' capacity to express or release eotaxin and RANTES. Upon appropriate cytokine stimulation, lung fibroblasts were also found to express messenger RNA for monocyte chemotactic protein (MCP)-3 and MCP-4 but not eotaxin-2. However, no ECA like MCP-3 or MCP-4 was detected. These observations suggest that the release of Th1 or Th2 cytokines in the lung tissue polarizes lung fibroblasts to produce either RANTES or eotaxin as major Eo attractants.

Expression of Th1 and Th2 Immunoregulatory Cytokines by Human Eosinophils

Besides cytotoxic mediators, human eosinophils can produce proinflammatory and anti–inflammatory cytokines, as well as growth factors and chemokines. The demonstration that eosinophils from patients could produce IL–5, IL–4 and IL–2 suggested their participation in the regulation of immune response. In the present work, we have examined the presence of Th1 (IFN–γ, IL–2) and Th2 (IL–4, IL–5, IL–10 and IL–13) cytokines in eosinophils from patients or donors by intracellular flow cytometry, and by immunocytochemistry. Whereas almost 100% eosinophils expressed intracellular IFN–γ, IL–2 and IL–10, the expression of IL–4, IL–5 and IL–13 is more variable and increased in patients versus donors. The differential release by eosinophils of Th2 versus Th1 cytokines is suggested both by the decrease of the intracellular content after culture, restricted to IL–4, IL–5 and IL–13, associated with an accumulation of IL–4 and IL–5 in the presence of Brefeldin. These results indicate that, through the preferential release of Th2 cytokines, human eosinophils can participate in the polarization of the immune response.

Effect of seasonal exposure to pollen on nonspecific interleukin-4, interleukin-5, and interferon-gamma in vitro release by peripheral blood mononuclear cells from subjects with pollinosis.

The immune response to environmental allergens depends on both genetic and environmental factors. Allergen exposure triggers the activation of allergen-specific Th2 cells in allergic patients, as well as increased Th2-type cytokine mRNA expression and eosinophil recruitment. Nevertheless, different patterns of release of cytokines could explain the heterogeneity of atopic response. In our study, 25 patients with pollinosis and 15 healthy donors were selected to characterize their release of Th2 (interleukin [IL]-4 and IL-5) and Th1 (interferon-gamma [IFN-gamma]) cytokines, both during and outside the pollen season. Peripheral blood mononuclear cells from patients and controls were isolated, cultured in the presence of phorbol-12-myristate-13-acetate plus ionomycine, and phytohemagglutinin (PHA), and cytokine release was assessed by titration in the supernatants. Both IL-4 and IL-5 showed higher levels during than outside the pollen season in pollinic patients (P<0.05) after nonspecific stimuli, whereas IFN-gamma levels were significantly lower during than outside the pollen season only after culture with PHA. Significant differences were not observed in the control group. Our results are consistent with the hypothesis that release of cytokines by peripheral blood mononuclear cells from patients with pollinosis depends on environmental exposure to sensitizing pollens, and that influence can be revealed by in vitro nonspecific stimulation. Nevertheless, the heterogeneity in results suggests that the use of mitogens to assess Th1/Th2 dominance may need careful evaluation.

Peptide-mediated regulation of allergen-specific immune responses

The exposure of susceptible individuals to environmental allergens induces enhanced levels of specific IgE antibodies, which bind to high affinity receptors on mast cells and basophils. Cross linking of receptor-bound IgE occurs, mediators are released and the immediate allergic reactions develop. Increased vascular permeability and the production of cytokines and chemokines cause polymorphonuclear granulocytes and lymphocytes to migrate into the site of allergic reactions and the late phase reaction is established. This illustrates that the cellular and cytokine networks activated in the pathogenesis of allergic inflammation are complex and involve many different cell types. However, the regulation of allergic immune responses in atopic individuals is mediated by cytokines characteristic of the Th2 functional phenotype (IL-4, IL-5, IL-10 and IL-13).1–5 In non-atopic individuals Thl cells, which produce IL-2 and IFN-γ, but not IL-4 or IL-5, are activated and regulate immune responses to allergens.6,7 However, from clonal analysis it is evident that Th0 cells, which have the potential to produce both Thl and Th2 cytokines, form a major component of the allergen specific repertoire in atopic individuals. Therefore, the ability to modify qualitative aspects of immune responses to allergens in atopic individuals by inhibiting the release of Th2 cytokines, and/or promoting the production of Thl cytokines may alter the clinical phenotype of some patients. The approach of immunotherapy mediated by peptides and designed to target allergen reactive CD4+ T cells are discussed in this report.

Blockade of CD49d (alpha4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma.

Immunized mice after inhalation of specific antigen have the following characteristic features of human asthma: airway eosinophilia, mucus and Th2 cytokine release, and hyperresponsiveness to methacholine. A model of late-phase allergic pulmonary inflammation in ovalbumin-sensitized mice was used to address the role of the alpha4 integrin (CD49d) in mediating the airway inflammation and hyperresponsiveness. Local, intrapulmonary blockade of CD49d by intranasal administration of CD49d mAb inhibited all signs of lung inflammation, IL-4 and IL-5 release, and hyperresponsiveness to methacholine. In contrast, CD49d blockade on circulating leukocytes by intraperitoneal CD49d mAb treatment only prevented the airway eosinophilia. In this asthma model, a CD49d-positive intrapulmonary leukocyte distinct from the eosinophil is the key effector cell of allergen-induced pulmonary inflammation and hyperresponsiveness.

T cell receptor V beta complementarity-determining region 1 peptide administration moderates immune dysfunction and cytokine dysregulation induced by murine retrovirus infection.

Murine AIDS, induced by LP-BM5 murine leukemia retrovirus infection, causes a progressive and profound immunodeficiency in female C57B1/6 mice. Previously, we reported that autoantibodies were elevated during the initiation phases of this murine retrovirus infection and bound peptide determinants corresponding to CDR1 of several TCR V beta-chains. Therefore, we designed studies to determine whether administration of a major autoimmunogenic TCR V beta CDR1 peptide before or after infection with LP-BM5 retrovirus would modulate retrovirus-induced dysregulation of T cell function. Administration of the TCR V beta CDR1 peptide before murine retrovirus infection significantly prevented its suppression of splenic NK cell activity, T and B cell proliferation, and monokine (IL-6 and TNF-alpha) and Th1 cytokine (IL-2 and IFN-gamma) release by splenocytes, and inhibited retrovirus-induced elevation of Th2 cytokine (IL-5 and IL-10). Similar data were obtained with peptide immunization 2 wk after murine retrovirus infection at 6 and 16 wk postinfection. However, delaying peptide immunization until severe suppression of T and B cell mitogenesis had occurred did not restore their functions. Immunization with TCR V beta peptide prevents development of retrovirus-induced immune dysfunction, which suggests a possible pathogenic role of autoreactive T cells as regulatory elements.