Th2 cells and cytokine networks in allergic inflammation of the lung

The cytokines released from Th2 and Th2-like cells are likely to be central to the pathophysiolgy of asthma and allergy, contributing to aberrant IgE production, eosinophilia and, perhaps, mucosal susceptibility to viral infection. IL-4 has emerged as a central target, not only for B cell IgE production, but also in the commitment of both CD4+ and CD8+ T cells to cells with Th2 effector function capable of secreting IL-5 resultlng in eosinophilic inflammation. In view of the central role of this cytokine and the evidence that glucocorticoids are unable to modify many IL-4 dependent effects, Th2 inhibitors may prove to be novel therapies for the treatment of bronchial asthma.     

IL-4 and IL-2 selectively rescue Th cell subsets from glucocorticoid-induced apoptosis.

It is well established that T cell maturation and activation are negatively regulated by a mechanism termed apoptosis. We now present evidence that glucocorticoids, known to possess immunosuppressive properties, cause apoptosis in mature Th cells, similarly to what has been reported for thymocytes. Th cells treated with the synthetic glucocorticoid dexamethasone show genome fragmentation into oligonucleosomal fragments, and proliferation of growth factor stimulated Th cells is inhibited by glucocorticoids. We show that IL-4 specifically rescues Th2 cells from dexamethasone-mediated apoptosis, whereas IL-2 and IL-1 are ineffective in these cells. However, IL-2 is the relevant rescue-factor of glucocorticoid-treated Th1 cells. The rescue induced by IL-4 and IL-2 is thought to be mediated by protein kinases (possibly protein kinase C), as evidenced by the fact that the protein kinase inhibitor H7 blocks the action of IL-4 and IL-2 in glucocorticoid-treated cells. Our in vitro data show that mature T cells can be protected by their own growth factors from the deleterious effects of the synthetic glucocorticoid dexamethasone, and suggest that specific interactions occur between lymphokines and naturally produced glucocorticoids in vivo, which may play a role in the regulation of the immune response.     

Direct and indirect inhibition of Th1 development by progesterone and glucocorticoids.

Progesterone may contribute to the maternal suppression of immunity to the fetus by modulating the Th1/Th2 balance. To clarify whether progesterone directly or indirectly affects T cell differentiation, we used two experimental systems with isolated T cells in vitro. In one system, isolated CD4+CD8+thymocytes differentiated into Th1 and Th2 by two pulse stimulations with defined combinations of ionomycin and PMA followed by the treatment with IL-12, IL-4, and IL-2. In the second system, functional differentiation was induced in purified naive CD4 T cells with cytokines and Abs to CD3 and CD28. In both systems, progesterone added with cytokines suppressed Th1 development at concentrations associated with pregnancy, but enhanced the development of IL-10-producing Th2 cells. Because IL-10 is known to inhibit APC production of IL-12, Th1 development may be also suppressed indirectly by progesterone. However, progesterone failed to enhance IL-10 production in the absence of IL-12. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 inhibited Th1 development and enhanced Th2 development, as did progesterone, indicating that p38 MAPK and extracellular signal-regulated kinase pathways are involved in Th1 development. However, the progesterone effects may not be simply due to a modulation of MAPK activities, because the inhibitor did not significantly affect the development of IL-10-producing cells in the presence or absence of progesterone. Glucocorticoids exerted effects similar to those of progesterone on Th1/Th2 development even at lower concentrations. These results suggest that progesterone as well as glucocorticoids directly inhibit Th1 development and enhance Th2 development.     

Differential regulation of Th2 and Th1 lung inflammatory responses by protein kinase C theta

In vitro and recent in vivo studies have identified protein kinase Ctheta (PKCtheta) as an important intermediate in signaling pathways leading to T cell activation, proliferation, and cytokine production. However, the importance of PKCtheta to many T cell-driven inflammatory responses has not been demonstrated. In this study we show that although PKCtheta is required for the development of a robust lung inflammatory response controlled by Th2 cells, it plays a lesser role in the development of a similar lung inflammatory response controlled by Th1 cells. PKCtheta-deficient mice were strongly compromised in generating Th2 cells and exhibited reduced airway eosinophilia and Th2 cytokine production in lungs. PKCtheta was required for the initial development of Th1 cells, with these cells exhibiting delayed kinetics of differentiation and accumulation. However, with recall Ag challenge via the airways, this defect was overcome, and lung infiltration and Th1 cytokine production were largely unimpaired in PKCtheta-deficient animals. These data suggest that PKCtheta can play roles in aspects of both Th2 and Th1 responses, but lung inflammation induced by Th2 cells is more dependent on this protein kinase than lung inflammation induced by Th1 cells.     

Exclusive Th2 primary effector function in spleens but mixed Th1/Th2 function in lymph nodes of murine neonates

Recent studies have shown that neonatal mice are competent to develop mature, Ag-specific Th1 function in situ. However, under many conditions, Th2 responses dominate in the neonate, while Th1 responses are more prevalent in adults. To compare further the immune responses of neonates and adults, we used the enzyme-linked immunospot method to measure the frequencies of primary Th1/Th2 effectors generated in situ in the spleens and lymph nodes. As assessed by the detection of IFN-gamma- or IL-4-producing cells, adults developed mixed Th1/Th2 responses in both organs. Neonatal lymph nodes contained mature frequencies of IFN-gamma- and IL-4-producing cells. In striking contrast, while mature frequencies of Th2 cells developed in neonatal spleens, virtually no IFN-gamma-secreting cells were detected. Exclusive Th2 function was observed in both BALB/c and C57BL/6 neonates, strains in which the Th2 and Th1 lineages, respectively, are favored in adults. Although Th1 effectors were virtually undetectable, the addition of rIL-12 boosted the frequency of IFN-gamma-secreting cells to adult levels. Therefore, Th1 effectors apparently developed in situ, but Th1 effector function either was not promoted or was inhibited upon subsequent exposure to the Ag in culture. Together, these results indicate that the quality of a primary Th response in neonates is strongly dependent on the site of initial Ag exposure; responses initiated in the lymph nodes are mixed Th1/Th2, whereas responses occurring in the spleen are heavily Th2 biased.     

Antigen-specific Th1 but not Th2 cells provide protection from lethal Trypanosoma cruzi infection in mice

Infection with Trypanosoma cruzi results in the development of both type 1 and type 2 patterns of cytokine responses during acute and chronic stages of infection. To investigate the role of Th1 and Th2 subsets of CD4(+) T cells in determining the outcome of T. cruzi infection in mice, we have developed T. cruzi clones that express OVA and have used OVA-specific TCR-transgenic T cells to generate OVA-specific Th1 and Th2 cells. BALB/c mice receiving 10(7) OVA-specific Th1 cells and then challenged with OVA-expressing T. cruzi G-OVA.GPI showed significantly lower parasitemia and increased survival in comparison to mice that received no cells. In contrast, recipients of OVA-specific Th2 cells developed higher parasitemias, exhibited higher tissue parasitism and inflammation, and had higher mortality than recipients of Th1 cells after infection with T. cruzi G-OVA.GPI. Mice receiving a mixture of both Th1 and Th2 OVA-specific cells also were not protected from lethal challenge. The protective effect of the OVA-specific Th1 cells was OVA dependent as shown by the fact that transfer of OVA-specific Th1 or Th2 cells failed to alter the course of infection or disease in mice challenged with wild-type T. cruzi. Immunohistochemical analysis of OVA-specific Th1 and Th2 cells at 4, 15, and 30 days postinfection revealed the persistence and expansion of these cells in mice challenged with T. cruzi G-OVA.GPI but not in mice infected with wild-type T. cruzi. We conclude that transfer of Ag-specific Th1 cells but not Th2 cells protect mice from a lethal infection with T. cruzi.     

Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.     

IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis.

Resistance to Leishmania major in mice is associated with the generation of distinct CD4+ Th subsets, termed TH1 and TH2. To define the factors contributing to the genesis of these Th cells, we first investigated when these subsets developed following L. major infection. Lymph node (LN) cells collected 3 days after infection of BALB/c mice secreted IL-4 and IL-5 in vitro, but little IFN-gamma, whereas LN cells from a resistant strain, C3H/HeN, secreted IFN-gamma and no IL-4 or IL-5. Cytokine production was eliminated in both cases by in vivo or in vitro depletion of CD4+ cells, but not after depletion of CD8+ cells. Similar responses were observed after inoculation of killed promastigotes or a soluble leishmanial Ag preparation. These data indicate that the development of Th1- and Th2-like responses can precede lesion formation and does not require a live infection. We next investigated whether IFN-gamma was important in the differentiation of Th1 and Th2 cells. C3H/HeN mice have previously been shown to be susceptible to leishmanial infection after treatment with anti-IFN-gamma. We confirmed this observation and found that the abrogation of resistance was associated with enhanced production of IL-4 and IL-5, and decreased production of IFN-gamma by cells taken from these mice. Conversely, LN cells from BALB/c mice inoculated with parasites plus IFN-gamma produced significantly higher levels of IFN-gamma, and decreased levels of IL-4 and IL-5, than mice infected with parasites alone. Finally, we determined if IFN-gamma might augment vaccine induced immunity. We found that s.c. immunization with soluble leishmanial Ag, the bacterial adjuvant, Corynebacterium parvum and IFN-gamma could protect mice against L. major infection, and that this protection was associated with induction of Th1 responses. From these data we conclude that levels of IFN-gamma at the time of infection or immunization dramatically alters the type of response elicited: high levels of IFN-gamma favor Th1 type responses, whereas low levels promote a Th2 response.     

Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines.

PGE2 is known to inhibit IL-2 and IFN-gamma production from Th cells and is widely viewed as a general immunosuppressant. However, PGE2 was found not to inhibit IL-4 production from Th2 clones, and IL-5 production from these clones was slightly enhanced. The same results were obtained with short term T cell lines, which indicates that the lack of inhibition of IL-4 and IL-5 production by PGE2 is a general phenomenon. PGE2 functions by increasing cAMP levels through activation of adenylate cyclase. Despite its failure to inhibit lymphokine release, PGE2 was capable of increasing cAMP levels in Th2 cells, and forskolin, a direct activator of adenylate cyclase, also did not inhibit IL-4 or IL-5 production. These data indicate that the failure of PGE2 to inhibit IL-4 and IL-5 production was not due to an inability of PGE2 to induce an increase in intracellular cAMP, and suggested instead that the expression of IL-4 and IL-5 in Th2 cells is insensitive to elevated cAMP levels. When Th0 clones were examined, PGE2 was again found to differentially affect IL-2 and IL-4 production in three of five clones tested. In two additional Th0 clones, both IL-2 and IL-4 production were inhibited. These data suggest that lymphokine production may be regulated on two different levels. First, Th1- and Th2-associated lymphokines may be differentially sensitive to intracellular signals such as cAMP. Second, T cell subsets may exist, including subsets of Th0 cells, with different signaling pathways. In addition, our data suggest that PGE2 may play an important role in regulating the development of a response dominated by Th1- or Th2-associated lymphokines.     

Cutting edge: CD94/NKG2 is expressed on Th1 but not Th2 cells and costimulates Th1 effector functions

Th1 and Th2 cells can be phenotypically distinguished by very few cell surface markers. To identify cell surface molecules that are specifically expressed on Th1 cells, we have generated a panel of mAbs that specifically bind the surfaces of murine Th1 but not Th2 cells. One of these Abs identified the NK cell receptor CD94 as a molecule also specifically expressed on the surface of Th1 cells. As in NK cells, CD94 is expressed on Th1 cells together with members of the NKG2 family of molecules, including NKG2A, C, and E. Cross-linking these receptors on differentiated Th1 cells in vitro costimulates proliferation and cytokine production with a potency similar to that obtained by cross-linking CD28. We propose that CD94/NKG2 heterodimers may costimulate effector functions of differentiated Th1 cells.     

Collaboration of Th1 and Th2 T cell clones in specific antibody responses: regulation of the IgM response to phosphorylcholine

Carrier (KLH)-specific type 1 T cell clones (Th1), which are defined by secretion of IL-2 and IFN-gamma but not IL-4, and type 2 (Th2) clones, which secrete IL-4, but not IL-2 or IFN-gamma, have been isolated and analyzed for their ability to collaborate in providing help for B cells to secrete phosphorylcholine-specific IgM antibodies. The resulting antibody responses exhibited a characteristic pattern suggesting two distinct regulatory interactions among the Th1, Th2, and B cells. At low doses of antigen, Th1 cells enhanced the helper function of the Th2 cells, an effect due primarily to IL-2. At high doses of antigen, Th1 cells or IFN-gamma inhibited Th2-dependent antibody responses. The inhibitory effect of Th1 or IFN-gamma affected primarily the hapten-carrier-linked portion of the response. The overall effect was a modulation of the antigen dose-response curve for antibody production, eliminating the sharp increases in dose response mediated by isolated T cell clones. The data suggest that collaborative interactions of Th1 and Th2 cells in antibody production may have important physiological consequences.     

Distinctly different sensitivity in the induction and reversal of anergy of Th1 and Th2 cells

T cell anergy is one of the mechanisms of immunological tolerance. We examined in this study the distinct responses of Th1 and Th2 cells to in vitro anergic stimulation using Th1 and Th2 cells from two strains of T cell receptor transgenic mice. Proliferation of the Th2 cells was difficult to suppress by anergic stimulation, while that of Th1 cells was significantly inhibited even by weak stimulation. However, IL-4 production by Th2 cells was definitely reduced by anergic stimulation, although the inhibition level of IL-4 was lower than that of IFN-gamma production by Th1 cells. We also examined the reversal of anergy in both subsets. While both the anergized Th1 and Th2 cells responded to IL-2 stimulation, only the anergy of the Th2 cells could be reversed. This result indicates that progression of the cell cycle was not sufficient for anergy reversal in Th1 cells. Our findings indicate that the induction and reversal of T cell anergy might be affected by the distinct signaling features of Th1 and Th2 cells.     

Murine Th1 and Th2 clones proliferate optimally in response to distinct antigen-presenting cell populations.

We recently have devised a method for the derivation of OVA-specific Th1 and Th2 clones from the same primed lymph node cell preparation. Using a panel of such cells, we have examined the ability of distinct APC populations to stimulate proliferation of Th1 and Th2 clones. Both subsets proliferated well in response to OVA in the presence of whole spleen cells. However, purified B cells stimulated optimal proliferation of Th2 clones, whereas adherent cells stimulated optimal proliferation of Th1 clones. The proliferative response of Th2 cells stimulated with spleen cells irradiated with 3300 rad was dramatically less than that observed in response to spleen cells treated with 1000 rad; Th1 clones responded similarly to spleen cells exposed to either irradiation dose. Differential activation of Th1 and Th2 clones did not correlate with MHC-restricting element, or susceptibility to inhibition by mAb directed against CD4 or LFA-1. Lymphokine production by each subset still occurred under conditions of suboptimal proliferation, suggesting that the appropriate Ag processing and presentation events had transpired. The same pattern of response was observed using a specific OVA peptide that does not require processing, suggesting that differential responsiveness of Th1 and Th2 clones to different APC populations is not a result of defective Ag processing. Neither rIL-1 nor rIL-6 restored optimal proliferation of either subset. Our results suggest that unique cofactors are necessary for the optimal proliferation of Th1 and Th2 clones, and that these cofactors are produced by specialized APC populations.     

Th1 and Th2 cytokine immunomodulation by gangliosides in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis, a classical model for multiple sclerosis, the cytokines provide the necessary signals to activate specific T cells for self-antigens. Gangliosides have multiple immunomodulatory activities, decreasing the lymphoproliferative responses and modulating cytokine production. Here, we tested the effects of gangliosides on the switching of Th1 to Th2 cytokine expression, in spleen cells obtained from Lewis rats during the acute phase of EAE, and after recovery from the disease. For this purpose, total RNA from spleen cells was isolated and submitted to RT-PCR to investigate Th1 (IL-2, TNF-alpha, and IFN-gamma) and Th2/Th3 (IL-10 and TGF-beta) cytokine gene expression. Results demonstrate that the group treated with gangliosides displays mild disease, with low expression of IFN-gamma mRNA and high TGF-beta mRNA expression. We conclude that the gangliosides may modulate Th1 cells by the synthesis of cytokines shifting the profile to the Th2/Th3 phenotype.     

Defining Th1 and Th2 immune responses in a reciprocal cytokine environment in vivo

The ability of committed Th1 and Th2 cells to function in altered cytokine environments is a central issue in autoimmune and immune-mediated diseases. Therefore, it is of interest to study the ability of Th1 or Th2 cells to expand and produce cytokine reciprocal environments in vivo. Using STAT4- and STAT6-deficient mice, we studied the expansion and cytokine production of Ag-specific Th1 or Th2 cells after transfer into Th1, Th2, or wild-type recipients. Our data show that these Th1 or Th2 cells proliferated and clonally expanded normally, regardless of the in vivo cytokine environment. These data have implications for the treatment of immune-mediated diseases by immunomodulatory agents that alter the cytokine milieu in vivo.     

Glucocorticoid effects on growth, and androgen receptor concentrations in DDT1MF-2 cell lines

The DDT1MF-2 smooth muscle tumor cell line contains receptors for and is differentially sensitive to androgens and glucocorticoids. Androgens stimulate and glucocorticoids inhibit growth. We now confirm that the latter involves the induction of a block in the G1 phase of the cell cycle. We have developed and characterized in vitro and in vivo a glucocorticoid resistant variant of this cell line, the DDT1MF-2-GR. Glucocorticoids specifically inhibit androgen induced androgen receptor augmentation in DDT1MF-2 cells, but not in the GR variant suggesting that growth inhibition is related to inhibition of androgen receptor augmentation. However, under optimal conditions for cell proliferation, when glucocorticoid inhibited growth is relieved by the exogenous addition of platelet derived growth factor, androgen receptor augmentation is still suppressed. Thus, androgen induced elevation in androgen receptor concentrations is not a prerequisite for cell proliferation. These results imply that in androgen responsive cells, although androgen stimulation of growth can be blocked by antagonism of androgen receptor mediated events, the antagonism can be bypassed by supplying the cells with exogenous growth factors. These results provoke speculation on how cells, which are dependent upon androgens for growth, become autonomous.     

Neonatal tolerance in the absence of Stat4- and Stat6- dependent Th cell differentiation

Neonatal tolerance to specific Ag is achieved by nonimmunogenic exposure within the first day of life. The mechanism that regulates this tolerance may provide the basis for successful organ transplantation and has recently been thought to be immune deviation from the inflammatory Th1 response to a Th2 response. To test the importance of Th2 cells in the establishment of neonatal tolerance, we examined neonatal tolerance in Stat4- and Stat6-deficient mice, which have reduced Th1 and Th2 cell development, respectively. Neonatal tolerance of both the T and B cell compartments in Stat4- and Stat6-deficient mice was similar to that observed in wild-type mice. Cytokine production shifted from a Th1 to a Th2 response in wild-type mice tolerized as neonates. In contrast, tolerance was observed in Stat6-deficient mice despite maintenance of a Th1 cytokine profile. These results suggest that cells distinct from Stat6-dependent Th2 cells are required for the establishment of neonatal tolerance.     

Th1/Th2 differentiation and cross-regulation

The T helper (Th) phenotypes, Th1/Th2, are acquired upon interaction of a naive T helper cell and an antigen presenting cell (APC). Naive T helper cells may differentiate into either phenotype, and the actual outcome is determined by the density and avidity of the antigenic determinants presented by the APC, and the APCs inherent costimulatory properties. Until recently it was thought that differentiation is further affected by cytokines. However, Murphy et al. (1996, J. Exp. Med. 183, 901) have demonstrated that the experimental results, formerly interpreted as Th1/Th2 differentiation, in effect comprise an observation of two consecutive processes. (i) An interaction between naive T cells and APC creates a mixture of mature cells irreversibly committed to Th1 or Th2 phenotype. (ii) Subsequent addition of regulatory cytokines, promotes expansion of one phenotype while suppressing the other. The consequent shift in the per culture production of marker cytokines mimics the appearance of a cellular phenotype switch. We present and analyse a mathematical model that extrapolates these experimental facts into systemic behavior during an immune response. Despite the fact that differentiation produces cells of Th1 and Th2 phenotypes with the same receptor specificity, our results indicate that competition for antigenic stimulation, mediated by the APCs, combines with cytokine mediated cross-suppression between phenotypes to yield a response that is eventually dominated by T helper cells that are uniform in both receptor specificity (clonotype) and in cytokine secretion phenotype.