T cell chemokine receptor expression in human Th1- and Th2-associated diseases

The interaction between chemokines and their receptors is an important step in the control of leukocyte migration into sites of inflammation. Chemokines also mediate a variety of effects independent of chemotaxis, including induction and enhancement of Th1- and Th2-associated cytokine responses. Recent studies have shown that human Th1 and Th2 clones, activated under polarizing conditions with polyclonal stimuli in vitro, display distinct patterns of chemokine receptor expression: Th1 clones preferentially express CCR5 and CXCR3, while many Th2 clones express CCR4, CCR8 and, to a lesser extent, CCR3. These differential patterns of chemokine receptor expression suggest a mechanism for selective induction of migration and activation of Th1- and Th2-type cells during inflammation and, perhaps, normal immune homoeostasis. Studies have begun to examine T cell chemokine receptor expression in vivo to determine the relevance of these in vitro observations to human Th1- and Th2-associated diseases. In this review, we critically examine recent reports of T cell chemokine receptor expression in human autoimmune disorders (multiple sclerosis and rheumatoid arthritis) and atopic disorders (allergic rhinitis and asthma) which are believed to arise from inappropriate Th1- and Th2-dominated responses, respectively.     

Granulocyte macrophage colony-stimulating factor-driven respiratory mucosal sensitization induces Th2 differentiation and function independently of interleukin-4

The development of T helper (Th)2 responses is a key step in the pathogenesis of asthma. Interleukin (IL)-4 is thought to be important, although not strictly necessary, for Th2 differentiation, although triggers of IL-4-independent Th2 polarization have not been identified. We examined whether IL-4 is necessary for Th2-polarized responses during granulocyte macrophage colony-stimulating factor (GM-CSF)-driven respiratory mucosal sensitization. Balb/c wild type (WT) or IL-4 knockout (4KO) mice were exposed to aerosolized ovalbumin (OVA) in the context of airway GM-CSF expression. We examined the extent of Th2 polarization using real-time quantitative polymerase chain reaction on lymph node mRNA, flow cytometric analysis of lung Th cells, and measurement of cells, cytokines, and immunoglobulins in bronchoalveolar lavage (BAL) and serum. GATA-3 and CCR3, -4, and -8 were expressed in the lymph nodes of WT and 4KO mice at similar levels, as were IL-5 and IL-13 levels in the BAL, T1/ST2 on lung Th cells, and BAL eosinophils after recall challenge. With the exception of immunoglobulin production, expression of GATA-3, CCR-3, -4, -8, IL-5, and T1/ST2, and the generation of blood eosinophilia, were intact in mice doubly deficient in both IL-4 and IL-13. We conclude that IL-4 is not required for the generation of Th2-polarized responses in the presence of GM-CSF.     

Assessment of chemokine receptor expression by human Th1 and Th2 cells in vitro and in vivo.

The preferential association of some chemokine receptors with human Th1 or Th2 cells has recently been reported. In this study, the expression of CCR3, CCR5, CXCR3, and CXCR4 were analyzed by flow cytometry in three distinct in vitro models of Th1/Th2 polarization, activated naive and memory T cells, and T-cell clones, in which the intracellular synthesis of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) and the surface expression of CD30 and LAG-3 were also assessed. Moreover, by using immunohistochemistry the in vivo expression of CCR3, CCR5, CXCR3, and CXCR4 was examined in the gut of patients suffering from Crohn's disease, a Th1-dominated disorder, and in the skin of patients suffering from systemic sclerosis, a Th2-dominated disorder. CCR5 and LAG-3 exhibited the same pathway of Th1 association, whereas CXCR3 did not discriminate between Th1- and Th2-dominated responses. On the other hand, CCR3 was found only occasionally in a small proportion of allergen-specific memory T cells with Th2/ThO profile of cytokine production in vitro. However, it was neither seen in Th2-polarized activated naive T cells nor in established Th2 clones and could be detected in vivo only on non-T cells. Finally, whereas CXCR4 expression was not limited to Th2 cells in vivo, it was markedly up-regulated by IL-4 and down-regulated by IFN-gamma in vitro. Thus, the results of this study confirm the existence of flexible programs of chemokine receptor expression during the development of Th1 and Th2 cells. However, caution is advised in interpreting these receptors as surrogate markers of a given type of effector response.     

Evidence for PI-3K-dependent migration of Th17-polarized cells in response to CCR2 and CCR6 agonists

IL-17-producing Th cells (Th17) are a distinct subset of effector cells that bridge the innate and adaptive immune system and are implicated in autoimmune disease processes. CD4(+) splenocytes from DO11.10 mice were activated with OVA peptide(323-339) and maintained under Th17 polarization conditions, resulting in significantly higher proportions of IL-17(+) T cells compared with nonpolarized (Th0) cells. Th17-polarizing conditions significantly increased the proportion of cells expressing the chemokine receptors CCR2, CCR6, and CCR9 when compared with Th0 cells. In contrast, there was a significant decrease in the proportion of cells expressing CXCR3 under Th17-polarizing conditions compared with nonpolarizing conditions. The respective chemokine agonists for CCR2 (CCL2 and CCL12), CCR6 (CCL20), and CCR9 (CCL25) elicited migration and PI-3K-dependent signaling events in Th17-polarized cells, thus indicating that all three receptors were functionally and biochemically responsive. Furthermore, postmigration phenotypic analysis demonstrated that the agonists for CCR2 and CCR6, but not CCR9, stimulated a modest enrichment of IL-17(+) cells compared with the premigration population. Pan-isoform inhibitors of PI-3K/Akt signaling prevented CCR2- and CCR6-mediated, polarized Th17 cell migration in a concentration-dependent manner. The unique chemokine receptor expression pattern of Th17 cells and their corresponding PI-3K-dependent migratory responses are important for understanding the pathogenesis of autoimmune diseases and may provide opportunities for the application of CCR2 and CCR6 antagonists and PI-3K isoform-selective inhibitors in defined inflammatory settings.     

United airways: circulating Th2 effector cells in an allergic rhinitis model are responsible for promoting lower airways inflammation

Background Allergic rhinitis (AR) and asthma often coexist and are referred to as ‘united airways’ disease. However, the molecular and cellular pathways that are crucially involved in the interaction between upper and lower airways remain to be identified. Objective We sought to assess whether and how AR exacerbates lower airway inflammation upon allergen challenge in mice. Methods We previously developed an intranasal ovalbumin (OVA)‐driven AR model, characterized by nasal eosinophilic inflammation, enhanced serum levels of OVA‐specific IgE and Th2 cytokine production in cervical lymph nodes. In OVA‐sensitized mice with or without AR, a lower airway challenge was given, and after 24 h, lower airway inflammation was analysed. Results We found that AR mice were more susceptible to eosinophilic inflammation following a lower airway OVA challenge than OVA‐sensitized controls. AR mice manifested increased numbers of eosinophils in bronchoalveolar lavage fluid and increased inter‐cellular adhesion molecule‐1 (ICAM‐1) expression on lung endothelium, when compared with OVA‐sensitized controls. Depletion of T cells in OVA‐challenged AR mice completely abrogated all hallmarks of lower airway inflammation, including enhanced IL‐5 and tissue eosinophilia. Conversely, adoptive transfer of Th2 effector cells in naïve animals induced lower airway eosinophilic inflammation after challenge with OVA. Blocking T cell recirculation during AR development by the spingosine‐1 analogue FTY720 also prevented lower airway inflammation including ICAM‐1 expression in AR mice upon a single lower airway challenge. Conclusion Our mouse model of ‘united airways’ disease supports epidemiological and clinical data that AR has a significant impact on lower airway inflammation. Circulating Th2 effector cells are responsible for lung priming in AR mice, most likely through up‐regulation of ICAM‐1. Cite this as: A. KleinJan, M. Willart, M. van Nimwegen, K. Leman, H. C. Hoogsteden, R.W. Hendriks and B.N. Lambrecht, Clinical & Experimental Allergy, 2010 (40) 494–504.     

IL-18 together with anti-CD3 antibody induces human Th1 cells to produce Th1- and Th2-cytokines and IL-8

Although IL-18 was initially regarded as a factor that enhances IFN-gamma production from Th1 cells, later studies revealed its potential to induce Th2 cytokine production from T cells, NK cells and basophils/mast cells. Very recently, we demonstrated that passively transferred memory phenotype Th1 cells induce airway inflammation and hyperresponsiveness in a host mouse by production of Th1-, Th2-cytokines, GM-CSF and chemokines, when the transferred cells are stimulated in the host mice with nasally administered Ag and IL-18. Moreover, IL-18 is suggested to contribute to asthma exacerbation in human patients. Therefore, it is important to determine whether human Th1 cells also have the potential to produce these soluble factors when stimulated with anti-CD3 and IL-18 in vitro. Here we demonstrated that only Th1 cells, but not Th2 cells, produce IFN-gamma, IL-13, GM-CSF and IL-8 after stimulation with anti-CD3 and IL-18. Furthermore, highly purified IFN-gamma-producing Th1 cells have the same potential. Thus, human Th1 cells may become very harmful cells, when stimulated with Ag and IL-18 in vivo, and produce IFN-gamma, IL-13, GM-CSF and IL-8, which in combination might induce severe inflammation such as airway inflammation.     

Lipopolysaccharide inhalation exacerbates allergic airway inflammation by activating mast cells and promoting Th2 responses

BACKGROUND: Bacterial infection occasionally exacerbates asthma, although the cellular and molecular mechanisms have not been well defined. An involvement of mast cells has been suggested, as lipopolysaccharides (LPS)-induced cytokine production from mast cells in vitro. OBJECTIVE: This study was undertaken to examine the effects of LPS inhalation on mast cell functions and allergen-specific immune responses in a murine model of asthma. METHODS: Female BALB/c mice or mast cell-deficient W/W(v) mice were immunized intraperitoneally with ovalbumin (OVA). Mice were challenged with aerosolized OVA or OVA with LPS daily from day 21 to day 24. Twenty-four hours after the last challenge, airway inflammation and OVA-specific immune responses were examined. Allergen-specific T cell responses were further analysed by adoptively transferring OVA-specific CD4(+) T cells. Expression of chemokines in the lung was also examined. RESULTS: LPS inhalation with OVA resulted in exacerbated airway infiltration, which was not evident in mast cell-deficient mice. IL-5 production by mast cells in the lung was enhanced by LPS inhalation. OVA-specific IgE production as well as proliferation, cytokine production and local infiltration of OVA specific T-helper lymphocytes type 2 (Th2) were also enhanced. Up-regulated expression of Th2- and/or eosinophil-attracting chemokines was observed in the lung of mice inhalated with LPS. CONCLUSIONS: LPS inhalation exacerbates airway inflammation, which is accompanied by mast cell activation and enhanced Th2 responses. These observations provide clues towards understanding the mechanisms of bacterial infection-induced exacerbation of the clinical features of asthma.     

Th1 and Th2 in human IVF pregnancy with allogenic fetus

PROBLEM: Positive obstetric outcome of allogenic in vitro fertilization (IVF) pregnancies makes interesting the subject of additional regulatory mechanisms after oocyte donation. METHOD OF STUDY: Eighty eight women: 23 donation of oocytes (DO), 33 IVF, 32 natural conception (NC) were studied in first trimester of pregnancy. Intracellular production of cytokines and chemokine receptors expression were studied by flow cytometry. RESULTS: Intracellular production of interferon-gamma (IFN-gamma), interleukin (IL)-4, tumor necrosis factor-alpha by CD4 T lymphocytes in DO women was higher than in IVF and NC women. Ratio IFN-gamma/IL-4 in DO was lower than in IVF. We found higher expression of chemokine receptor CCR4 but not CXCR3 on CD4 T cells in DO compared with IVF and NC. Ratio CXCR3/CCR4 in DO was lower than in NC. CONCLUSION: Hyperactivation of T helper 1 (Th1) and T helper 2 (Th2) by allogenic fetus is specific for DO pregnancy. Preferable activation of Th2 and relative suppression of Th1 chemokine expression reflect additional regulatory counteractive mechanism(s).     

Switch in Chemokine Receptor Phenotype on Memory T Cells without a Change in the Cytokine Phenotype

Th1 and Th2 cells as defined by their cytokine profile are associated with the expression of the chemokine receptors CCR5 and CCR3, respectively. In committed human memory Th1 cells the cytokine profile is irreversibly expressed. However, it is not known if the chemokine receptor phenotypes of Th1 and Th2 cells are permanently associated to the cytokine profile or if it can be changed. To analyze the possibility of inducing a switch in chemokine receptor phenotype on memory Th cells we used differentiated memory Th cells isolated from synovial tissue (ST) samples of patients with rheumatoid arthritis (RA). Freshly isolated T cells, T-cell lines and T-cell clones from these tissues were manipulated with Th1 (interleukin (IL)-12 + anti IL-4) or Th2 (IL-4 + anti IL-12) inducing conditions. The surface expression of CCR5 and CCR3 was analyzed by flowcytometry and interferon (IFN)-gamma and IL-4 production by ELISA. A Th1-inducing cytokine environment increased the expression of CCR5 in Th1 cells and induced the expression of CCR5 in Th2 cells as compared to culture condition with only IL-2. Induction of CCR5 expression on Th2 clones was associated with secretion of some IFN-gamma. Moreover, the Th2-associated chemokine receptor CCR3 could be expressed on both Th1-dominant cell lines, and clones of Th1 and Th0 type after culture conditions with IL-4. This expression of CCR3 was associated with a reduced IFN-gamma production, but no IL-4 production could be induced. The IL-4-treated Th1 clones had a reduced migratory capacity against chemokines produced by ST cells compared to nonmanipulated T-cell clones. In contrast, the same IL-12-treated Th1 clones showed an increased migratory potential. Induction of the Th2-associated marker CCR3 on memory Th1 cells demonstrates that a change in chemokine receptor phenotype related to the Th2 type can be induced on terminally differentiated Th1 cells, without a change in the cytokine profile.     

Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization

It has been postulated that low-level interleukin (IL)-12 production of antigen-presenting cells is associated with the risk of developing atopic asthma. To study the relationship between IL-12 production capacity of dendritic cells (DCs) and development of T helper type 2 (Th2) responses in the lung, we genetically engineered DCs to constutively overexpress bioactive IL-12. Retrovirally mediated overexpression of IL-12 in DCs strongly polarized naive ovalbumin (OVA)-specific CD4+ T cells toward Th1 effector cells in vitro. After intratracheal injection, OVA-pulsed IL-12-overexpressing DCs failed to induce Th2 responses in vivo and no longer primed mice for Th2-dependent eosinophilic airway inflammation upon OVA aerosol challenge, readily observed in mice immunized with sham-transfected, OVA-pulsed DCs. Analysis of a panel of cytokines and chemokines in the lung demonstrated that the lack of Th2 sensitization was accompanied by increased production of the Th1 cytokine interferon-gamma (IFN-gamma), chemokines induced by IFN-gamma, and the immunoregulatory cytokine IL-10. When Th2 priming was induced using OVA/alum prior to intratracheal DC administration, DCs constitutively expressing IL-12 were no longer capable of preventing eosinophilic airway inflammation and even enhanced it. These data show directly that high-level expression of IL-12 in DCs prevents the development of Th2 sensitization. Enhancing IL-12 production in DCs should be seen as a primary prevention strategy for atopic disorders. Enhancing IL-12 production in DCs is less likely to be of benefit in already Th2-sensitized individuals.     

Selective recruitment of CCR4-bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine

Helper T cells are classified into Th1 and Th2 subsets based on their profiles of cytokine production. Th1 cells are involved in cell-mediated immunity, whereas Th2 cells induce humoral responses. Selective recruitment of these two subsets depends on specific adhesion molecules and specific chemoattractants. Here, we demonstrate that the T cell-directed CC chemokine thymus and activation-regulated chemokine (TARC) was abundantly produced by monocytes treated with granulocyte macrophage colony stimulating factor (GM-CSF) or IL-3, especially in the presence of IL-4 and by dendritic cells derived from monocytes cultured with GM-CSF + IL-4. The receptor for TARC and another macrophage/dendritic cell-derived CC chemokine macrophage-derived chemokine (MDC) is CCR4, a G protein-coupled receptor. CCR4 was found to be expressed on approximately 20% of adult peripheral blood effector/memory CD4+ T cells. T cells attracted by TARC and MDC generated cell lines predominantly producing Th2-type cytokines, IL-4 and IL-5. Fractionated CCR4+ cells but not CCR4- cells also selectively gave rise to Th2-type cell lines. When naive CD4+ T cells from adult peripheral blood were polarized in vitro, Th2-type cells selectively expressed CCR4 and vigorously migrated toward TARC and MDC. Taken together, CCR4 is selectively expressed on Th2-type T cells and antigen-presenting cells may recruit Th2 cells expressing CCR4 by producing TARC and MDC in Th2-dominant conditions.     

Dominance of CCL22 over CCL17 in induction of chemokine receptor CCR4 desensitization and internalization on human Th2 cells

Chemokines and their receptors play a pivotal role in controlling T cell trafficking in immunity and inflammation. Two chemokines, CCL17 and CCL22, activate the chemokine receptor CCR4, expressed on functionally distinct subsets of T cells: cutaneous leukocyte-associated antigen (CLA)+ skin-homing, T helper (Th) 2, and CD25+ T suppressor cells. Here, we compared the ability of CCL17 and CCL22 to promote CCR4 internalization as a mechanism of regulation of receptor function on human Th2 cells. We report that CCL22 is a potent and rapid inducer of CCR4 internalization, while CCL17 is not. CCR4 internalization does not require G protein coupling, while being dependent on lipid rafts integrity and clathrin-coated pits functionality. Cell surface disappearance of CCR4 is rapidly reversed upon removal of exogenous ligand by virtue of receptor recycling. CCR4 internalization leads to a loss of functional responsiveness, while recovery of surface expression leads to re-acquisition of chemotactic sensitivity of Th2 cells. The differential CCR4 desensitization and internalization reported here and the distinct expression patterns of CCL17 and CCL22 observed in vivo suggest that while CCL17 may act first on CCR4 at the endothelial surface to promote vascular recognition, CCL22 could subsequently engage the receptor within the tissue microenvironment to guide cellular localization.     

Inhibition by IL-12 and IFN-alpha of I-309 and macrophage-derived chemokine production upon TCR triggering of human Th1 cells

Th1 and Th2 cells, which produce distinct sets of cytokines, differentially express several chemokine receptors that may regulate their tissue-specific localization. Although the expression pattern and regulation of chemokines are likely to play a critical role in many immunopathological processes, they remain largely unknown. Here, we investigated the requirements for Th1 and Th2 cells to produce the Th2 cell-attracting chemokines thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and I-309. TCR triggering of Th1 and Th2 cells leads to production of MDC and I-309 (CCR4 and CCR8 ligands, respectively), whereas TARC (CCR4 ligand) is selectively produced by Th2 cells. Secretion of these chemokines appears to be independent of endogenous production of IL-4 and IFN-gamma. IL-12 and IFN-alpha, cytokines that promote the differentiation of human Th1 cells, selectively inhibit secretion and mRNA expression of MDC and I-309 by Th1 cells. Suppression of I-309 secretion results in a decreased chemotactic effect on L1.2 cells transfected with human CCR8, indicating that IL-12 and IFN-alpha may inhibit the recruitment of CCR8-expressing cells such as Th2 cells. The inhibition of Th2 cell-attracting chemokines MDC and I-309 illustrates a novel mechanism by which IL-12 and IFN-alpha could promote and maintain an ongoing Th1 response.     

Type 1 T-cell responses in chlamydial lung infections are associated with local MIP-1α response

Chemokines and their receptors are important mediators of leukocyte trafficking and recruitment and sometimes work as modulators of T-cell responses during infections and inflammation. Modulating the biological activity of chemokines has been found to influence the course of diseases. However, little is known about the role of chemokine responses during chlamydial lung infections. We therefore analyzed the dynamics of multiple chemokines, which are frequently associated with type 1 (Th1) T cell immune responses, and their receptors for their expression in the lungs during Chlamydia muridarum (Cm) infections. We also examined the relationship between chemokine responses and the development of Th1 responses as well as the clearance of infection. Our results showed that in parallel with the high levels of gamma interferon (IFN-γ) and IL-12 production in the lungs and draining lymph nodes, and the expansion of IFN-γ-producing CD4 and CD8⁺ T cells, the production of the cell-related chemokines RANTES, IFN-γ-inducible protein-10 (IP-10) and macrophage inflammatory protein-1α (MIP-1α) and their receptor CCR1 was elevated in the lung tissues after infection. Interestingly, in a later phase of infection, the expression of RANTES and IP-10 remained elevated but the expression of MIP-1α and CCR1 decreased to a low level, which suggests a closer association with the pattern of Th1 cytokine responses in the process of infection. These results suggest a close association between the MIP-1α response and the Th1-type T-cell responses in chlamydial lung infections.