Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells.

Airway inflammation associated with asthma is characterized by massive infiltration of eosinophils, mediated in part by specific chemoattractant factors produced in the lung. Allergen-specific Th2 cells appear to play a central role in asthma; for example, adoptively transferred Th2 cells induced lung eosinophilia associated with induction of specific chemokines. Interestingly, Th2 supernatant alone administered intranasally to naive mice induced eotaxin, RANTES, monocyte-chemotactic protein-1, and KC expression along with lung eosinophilia. We tested the major cytokines individually and found that IL-4 and IL-5 induced higher levels of macrophage-inflammatory protein-1alpha and KC; IL-4 also increased the production of monocyte-chemotactic protein-1; IL-13 and IL-4 induced eotaxin. IL-13 was by far the most potent inducer of eotaxin; indeed, a neutralizing anti-IL-13 Ab removed most of the eotaxin-inducing activity from Th2 supernatants, although it did not entirely block the recruitment of eosinophils. While TNF-alpha did not stimulate eotaxin production by itself, it markedly augmented eotaxin induction by IL-13. IL-13 was able to induce eotaxin in the lung of JAK3-deficient mice, suggesting that JAK3 is not required for IL-13 signaling in airway epithelial cells; however, eosinophilia was not induced in this situation, suggesting that JAK3 transduces other IL-13-mediated mechanisms critical for eosinophil recruitment. Our study suggests that IL-13 is an important mediator in the pathogenesis of asthma and therefore a potential target for asthma therapy.

Mouse IL-13 enhances antibody production in vivo and acts directly on B cells in vitro to increase survival and hence antibody production.

IL-13, a Th2 cytokine, exhibits similar functions to IL-4 in stimulating proliferation and class switching of human B cells. Although mouse B cells were reported to be unresponsive to IL-13, we now show that IL-13 directly stimulates mouse B cells, causing extended survival and higher Ab levels. Recombinant mouse IL-13 was administered via osmotic pump during immunization of BALB/c mice with chicken RBCs. IL-13 treatment enhanced not only the plasma levels of total IgG1, IgG2a, and IgG2b but also Ag-specific Ig levels. To examine whether IL-13 acted directly on mouse B cells, B220+ B cells were cultured with fixed, anti-CD3-activated Th2 clones. Production of IgM and IgG1 was enhanced moderately by IL-13 and strongly by IL-4. Anti-CD40-stimulated sIgD+ mouse B cells also responded to IL-13 by producing increased levels of IgM, and to a lesser extent IgG1, IgG2a, IgG2b, and IgG3. No evidence was found for IL-13-induced class switching. Mouse B cells were stimulated directly rather than indirectly via contaminating cells, as IL-13 increased the numbers of both total and Ab-secreting B cells in aliquots of 100 sIgD+ B cells (>99.5% pure) stimulated with anti-CD40 Ab. Stimulation of B cells by IL-13 was unaffected by the addition of anti-IL-4 to the cultures. In contrast to IL-4, IL-13 did not increase CD23 expression or B cell proliferation as measured by dilution of an intracellular fluorescence label. Collectively, these data indicate that IL-13 can enhance mouse B cell Ab production by increasing survival of the B cells.

Cytokine-deficient CD8+ Tc1 cells induced by IL-4: retained inflammation and perforin and Fas cytotoxicity but compromised long term killing of tumor cells.

After antigenic stimulation, naive CD8+ T cells differentiate into cytotoxic Tc1 cells secreting the cytokines IL-2, IFN-gamma, and TNF, which aid their proliferation and effector functions. We have previously shown that IL-4 acts directly on differentiated Tc1 cells to impair subsequent Con A-induced IL-2 production. As IL-4 may be produced in the vicinity of Tc1 cells during normal immune responses, we have further analyzed the short and long term functions of IL-4-treated Tc1 cells. We now show that these cells also have a defect in the synthesis of IFN-gamma, TNF, and IL-10 in response to antigenic stimulation. IL-2 synthesis was the most sensitive, as stimulation of IL-4-treated Tc1 cells with higher numbers of APCs partially restored IFN-gamma, TNF, and IL-10, but not IL-2, synthesis. Injection of allo-specific Tc1 cells into mice expressing the target Ag revealed reduced cytokine synthesis in vivo by IL-4-treated Tc1 cells. Loss of cytokine synthesis did not impair the short term effector functions of Tc1 cells, as they induced adoptively transferred delayed type hypersensitivity in recipient mice and retained both perforin- and Fas-dependent cytolytic mechanisms in vitro. Long term coculture of tumor targets and tumor-specific Tc1 cells indicated that normal Tc1 cells proliferated and killed tumor cells, whereas IL-4-treated Tc1 cells failed to proliferate and hence were unable to curtail the proliferation of tumor cells. These results suggest that IL-4 synthesis in vivo would not affect immediate effector functions of differentiated Tc1 cells, but would compromise immunity by reducing their long term functional capability.

CD8Tc1 and Tc2 cells secrete distinct cytokine patterns in vitro and in vivo but induce similar inflammatory reactions.

Naive CD8 T cells, similar to CD4 T cells, can differentiate into at least two subsets of cytolytic effector cells with distinct cytokine patterns: T cytotoxic-1 (Tc1) cells secrete a Th1-like cytokine pattern, including IL-2 and IFN-gamma; and Tc2 cells produce Th2 cytokines, including IL-4, IL-5, and IL-10. As CD4 Th1 cells induce delayed-type hypersensitivity (DTH) more effectively than Th2 cells, we tested the potential ability of Tc1 and Tc2 cells to induce DTH. Allospecific Tc1 or Tc2 cells were injected into the footpads of naive mice expressing the target Ag. Tc1 and Tc2 cells induced comparable levels of Ag-specific footpad swelling with similar kinetics. They also induced similar levels of footpad edema and similar infiltration of macrophages and neutrophils. However, Tc2 cells induced slightly more eosinophil infiltration. Analysis of footpad extracts showed that Tc1 and Tc2 cells retained their distinct in vitro cytokine profiles in the injected footpads. These results suggest that both Tc1 and Tc2 cytokines can be associated with the DTH reaction induced by CD8 T cells. Perforin-deficient Tc1 or Tc2 cells also induced DTH, although at lower levels, suggesting that perforin-mediated cytotoxicity of CD8 T cells is not essential for CD8-induced DTH. Thus, despite their distinct cytokine profiles in vitro and in vivo, Tc1 and Tc2 cells induce similar DTH reactions.

Functions of CD8 T-cell subsets secreting different cytokine patterns.

CD8+ T cells can differentiate into two effector phenotypes, Tc1 and Tc2, secreting different cytokine patterns. Both subsets are cytotoxic via the perforin and Fas pathways, and both kill resting and activated B cells, ruling out the possibility of cognate help, although Tc2 cells may provide bystander help. Both subsets induce inflammation with similar cellular infiltrates. Tc1 cytokine synthesis is limited by two mechanisms--IL-4 induces a permanent deficiency in cytokine secretion, and rapid killing of target cells limits CD8+ T-cell activation and cytokine production. These multiple CD8 T-cell activities provide a versatile set of immune functions.

Cytotoxicity and weak CD40 ligand expression of CD8+ type 2 cytotoxic T cells restricts their potential B cell helper activity.

Naive CD8+ T cells differentiate into distinct cytokine-secreting subsets: T helper (Th)1-like cytotoxic T cells (Tc1) and Th2-like Tc2. Although Th2 cells provide strong B cell help, we show that Tc2 cells secreting the same cytokines provide only modest B cell help for IgM production, and only when large numbers of B cells were stimulated with small numbers of Tc2 cells. Lack of effective B cell help by Tc2 cells was attributable partly to their cytotoxicity towards B cells. Both Tc1 and Tc2 cells killed small resting B cells mainly by a perforin-dependent mechanism. In contrast to normal Tc2 cells, perforin-deficient Tc2 cells failed to kill small resting B cells and induced IgM and IgG1 production, although their B cell help was significantly lower than that mediated by Th2 cells. This may be partly attributable to the ability of Tc2 but not Th2 cells to kill activated B cells even in the absence of perforin. Plate-bound anti-CD3 antibodies inhibited Tc2 killing of B cells and induced substantial immunoglobulin production. Additionally, Tc1 and Tc2 cells failed to express CD40 ligand (CD40L), whereas Th1 and Th2 cells expressed high levels of CD40L. Stimulation of Tc1 and Tc2 cells with plate-bound anti-CD3 antibodies for extended periods resulted in low-level expression of CD40L. Proliferation of small resting B cells correlated with immunoglobulin production: proliferation was promoted strongly by Th1 and Th2, weakly by normal Tc1 and Tc2, and moderately by perforin-deficient Tc1 and Tc2 cells. Thus, Tc2 cells may not contribute significantly to cognate B cell help during normal responses.

Differentiation and functions of T cell subsets.

The Tc1 and Tc2 subsets of CD8+ T effector cells secrete different patterns of cytokines, but have similar functions, including perforin- and Fas-dependent cytotoxicity, and induction of delayed type hypersensitivity (DTH) reactions involving oedema and granulocytic infiltration. The characteristic cytokines of Tc1 (gamma-interferon) and Tc2 (interleukins 4 and 5) are expressed in vivo during the DTH reaction. Tc1 cells that are deficient in cytokine synthesis also induce similar levels of DTH, supporting the lack of correlation between CD8+ T cell cytokine patterns and DTH. CD8+ T cells often produce lower cytokine levels than CD4 cells because the CD8 cells kill their antigen-presenting cells before full stimulation can occur. This effect can be counteracted by increasing the frequency of stimulation, or using perforin-deficient T cells. A multiparameter analysis of cytokine effects on CD8+ T cell differentiation has been initiated, on the basis of the principle that normal immune responses involve complex cytokine mixtures. All combinations of seven cytokines were tested. In some combinations, the combined effect could not have been predicted from individual cytokine functions. Conditions were identified in which each of interleukins 4, 10 and 12 could have opposite effects on CD8+ T cell differentiation.

Both CD4+ and CD8+ T-cells in syngeneic islet grafts in NOD mice produce interferon-gamma during beta-cell destruction.

Syngeneic pancreatic islet grafts in diabetic NOD mice are infiltrated by mononuclear leukocytes, beta-cells are selectively destroyed, and autoimmune diabetes recurs. This model was used to identify islet graft-infiltrating mononuclear leukocytes associated with beta-cell destruction and diabetes recurrence. We compared cell surface antigen and cytokine-producing phenotypes of mononuclear leukocytes in islet grafts from NOD mice that were protected from diabetes recurrence by complete Freund's adjuvant (CFA) administration (beta-cell nondestructive insulitis) and in islet grafts from control phosphate-buffered saline (PBS)-injected NOD mice (beta-cell destructive insulitis). Islet grafts from CFA-injected mice contained fewer CD4+ and CD8+ cells and more B cells; also fewer interferon gamma (IFN-gamma), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-alpha)-positive cells and more IL-4 and IL-10 positive cells. By performing two-color immunostaining of cell surface antigens and intracellular IFN-gamma, we found that IFN-gamma positive cells in islet grafts from CFA- and PBS-injected mice were approximately equally divided between CD4+ and CD8+ T-cell subsets. Also, the frequencies of both CD4+ IFN-gamma + and CD8+ IFN-gamma + cells were decreased in islet grafts from CFA-injected mice. These findings suggest that destruction of beta-cells in syngeneic islets transplanted into NOD mice is promoted by cells producing Th1-type cytokines (IFN-gamma, IL-2, and TNF-alpha) and prevented by cells producing TH2-type cytokines (IL-4 and IL-10). Furthermore, both CD4+ and CD8+ IFN-gamma-producing T-cells in the islet grafts appear to be involved in beta-cell destruction and diabetes recurrence.

Interleukin 10 modulates ion transport in rat small intestine.

BACKGROUND & AIMS: It has been proposed that the anti-inflammatory cytokine interleukin (IL)-10 might be an effective therapeutic agent in the treatment of inflammatory bowel disease. This study examined the effects of human recombinant IL-10 on ileal sodium and chloride transport in Sprague-Dawley rats. METHODS: Unidirectional fluxes of sodium and chloride and tissue electrical parameters were measured under voltage-clamped conditions in ussing chambers. Intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) were measured in isolated enterocytes. RESULTS: Jejunal and ileal tissue responded to serosal addition of IL-10 with a transient decrease in short-circuit current reflecting an IL-10-induced increase in net sodium and chloride absorption because of an increase in mucosal to serosal ion movement. The IL-10-induced absorption was not prevented by tetrodotoxin but did show tachyphylaxis. IL-10 reversed, or markedly attenuated, forskolin- and carbachol-induced net chloride secretion. The effects of IL-10 on net secretion were accompanied by a reduction in forskolin-stimulated cAMP levels and a decrease in basal cAMP levels. An additional effect of IL-10 was its induction of bicarbonate secretion only in the presence of secretagogues. CONCLUSIONS: This study shows that IL-10 enhances intestinal electroneutral sodium and chloride absorption, inhibits stimulated chloride secretion, and under some secretory conditions stimulates bicarbonate secretion.

Perforin and Fas killing by CD8+ T cells limits their cytokine synthesis and proliferation.

During an immune response, effector CD8+ T cells can kill infected cells by the perforin-dependent pathway. In comparison to CD4+ T cells, which are major sources of cytokines, normal CD8+ T cells produced less interleukin 2 and interferon gamma, and proliferated less vigorously after antigenic stimulation. Killing of target cells was a major cause of these reduced responses, since perforin-deficient CD8+ T cells showed substantially increased cytokine synthesis and proliferation. Cytotoxicity by the alternate Fas pathway also resulted in self-limitation of CD8+ T cell cytokine synthesis. This relationship between cytotoxicity and cytokine synthesis may regulate CD8+ T function in different phases of an immune response.

Continuous administration of Il-13 to mice induces extramedullary hemopoiesis and monocytosis.

IL-13, a recently identified Th2 cytokine, shares some, but not all, IL-4 functions, including inhibition of monocyte and macrophage activation, stimulation of human B cells, and induction of growth and differentiation of mouse bone marrow cells in vitro. We have now tested the in vivo effects of recombinant mouse IL-13 (rIL-13) from stably transfected, high expressing BW5147 thymoma cells. After purification by anion exchange chromatography, rIL-13 was administered in the peritoneal cavity of BALB/c mice via osmotic pump for 7 days. Spleens from the rIL-13-treated mice were significantly enlarged compared with control spleens due to increased cellularity. In particular, increased numbers of immature erythroblasts and megakaryocytes were observed in splenic sections after rIL-13 treatment. Spleen cells from rIL-13-treated mice showed greatly increased responsiveness in vitro to recombinant forms of mouse IL-3, mouse granulocyte-macrophage CSF, or human CSF-1 and, to a lesser extent, to mouse IL-4 or IL-13. Moreover, the rIL-13-treated mice also showed significant increases in CFU-E, CFU-C, and erythroid burst colonies in the spleen, further indicating the presence of increased numbers of hemopoietic precursors. Hematologic analyses indicated that rIL-13 treatment induced slight anemia and striking monocytosis. Finally, spleen cells from rIL-13-treated mice produced significantly more IL-6 upon LPS stimulation. Interestingly, the strong Th2 response induced by Nippostrongylus brasiliensis infection was also accompanied by an increase in hemopoietic precursor frequencies in the spleen. Collectively, these data indicate that exogenous rIL-13 induces extramedullary hemopoiesis in mice and suggest that endogenous IL-13 may contribute to replenishment of effector cells during strong Th2 responses.

The expanding universe of T-cell subsets: Th1, Th2 and more.

Since their discovery nearly ten years ago, T helper 1 (Th1) and Th2 subsets have been implicated in the regulation of many immune responses. In this article, Tim Mosmann and Subash Sad discuss the increasing number of T-cell subsets defined by cytokine patterns; the differentiation pathways of CD4+ and CD8+ T cells; the contribution of other cell types to these patterns; and the cytokine interactions during infection and pregnancy.

Pregnancy impairs resistance of C57BL/6 mice to Leishmania major infection and causes decreased antigen-specific IFN-gamma response and increased production of T helper 2 cytokines.

Resolution of cutaneous leishmaniasis in infected mice is associated with a polarized Th1 immune response by the host, whereas maternal immune responses during pregnancy appear to be biased toward humoral (Th2) and away from cell-mediated (Th1) responses. The objective of this study was to evaluate whether the putative Th2 bias in pregnant C57BL/6 mice would impair their normal ability to mount a curative Th1 response against Leishmania major infection. Pregnant C57BL/6 mice developed larger cutaneous lesions that showed no signs of resolution up to 70 days after infection. The infection appeared to be contained but not cured, as the footpad lesion remained stable, neither decreasing (as in normal C57BL/6 mice) nor showing uncontrolled expansion leading to death (as in susceptible mouse strains such as BALB/c). The number of parasites harvested from the footpads of pregnant mice was markedly higher than controls throughout the course of infection. The increased severity of infection in pregnant mice was accompanied by reduced IFN-gamma and increased IL-4, IL-5, and IL-10 production by spleen and popliteal lymph node cells stimulated in vitro with Leishmania Ags. Furthermore, IgG1 was elevated in the serum of pregnant mice as opposed to an increase of IgG2a in infected but nonpregnant controls. These observations support the existence of a bias toward Th2 cytokine expression during pregnancy and suggest that these cytokines effectively down-regulate the course of a normal Th1 response against a parasite infection in the periphery.

T helper 1 response against Leishmania major in pregnant C57BL/6 mice increases implantation failure and fetal resorptions. Correlation with increased IFN-gamma and TNF and reduced IL-10 production by placental cells.

Maternal immune responses can influence fetal survival and several cytokines have harmful or protective effects on pregnancy. The Th1 cytokines IFN-gamma and IL-2 can cause fetal loss, whereas the Th2 cytokine IL-10 is protective. However, infections such as leishmaniasis show the opposite pattern: resistance is associated with the preferential mounting of a Th1 response, whereas a Th2 response exacerbates the disease. We therefore asked whether the curative Th1 response against Leishmania major in genetically resistant C57BL/6 mice, would compromise concurrent pregnancy. The number of resorptions as assessed by uterine scars was significantly increased in infected C57BL/6 mice and this was associated with a decreased production by placental cells of the Th2 cytokines IL-4 and IL-10 and increased production of IFN-gamma and TNF. Interestingly, the frequency of pregnancy failure before implantation in C57BL/6 mice was also substantially increased. In contrast to C57BL/6 mice, early infection did not reduce implantations in BALB/c mice that mount a Th2 anti-L. major response and succumb to infection. For both resorptions and implantations, there appeared to be a short period early in infection that was detrimental to pregnancy, followed by a period with lesser effects, and a later period that again induced higher resorptions or pre-implantation losses. These results suggest that a beneficial anti-parasite Th1 response can adversely affect pregnancy outcome. Furthermore, Th1 cytokines may be deleterious for not only placental maintenance but also preimplantation events.

Interleukin (IL) 4, in the absence of antigen stimulation, induces an anergy-like state in differentiated CD8+ TC1 cells: loss of IL-2 synthesis and autonomous proliferation but retention of cytotoxicity and synthesis of other cytokines.

Naive T cells in the periphery mainly secrete interleukin (IL) 2 upon activation. After stimulation in the presence of appropriate costimulators, both CD4+ and CD8+ T cells differentiate into effector cells secreting distinct T helper (Th) 1- and Th2-like cytokine patterns. Subsequent to differentiation, both CD4+ (Th1 and Th2) and CD8+ (TC1 and TC2) cells are stable and cannot be induced to differentiate into the opposite pattern or revert to the naive cytokine secretion pattern. We now show that IL-4 caused committed TC1 bulk populations or clones to lose the ability to synthesize IL-2. The cells retained the ability to secrete interferon (IFN) gamma, granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor, did not synthesize any Th2 cytokines, and did not alter cell surface marker expression. IL-4 rapidly inhibited IL-2-synthesizing ability in the absence or presence of antigen-presenting cells, thus demonstrating that IL-4 acted directly on TC1 cells. The defect in IL-2 synthesis could not be reversed by subsequent stimulation with potent antigen-presenting cells in the presence of IL-2 and anti-IL-4, or with anti-CD3 plus anti-CD28 antibodies. Both IL-2+ and IL-2- TC1 cells were strongly cytotoxic toward allogeneic but not syngeneic targets. However, IL-2- TC1 cells were unable to proliferate unless exogenous IL-2 was provided. TC1 cells that lose IL-2 synthesis but retain IFN-gamma synthesis and cytotoxicity may be similar to the "anergic" cells induced by stimulation of CD4+ or CD8+ cells in the absence of costimulators. These results suggest that during a mixed type 1/type 2 response in vivo, IL-4 may induce the IL-2+ TC1-->IL-2-TC1 conversion, and thus curtail the expansion of the TC1 response without impairing short-term effector function.

Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines.

Alloantigen-stimulated CD8+ mouse spleen cells, either spontaneously or in the presence of IL-12 or IFN gamma plus anti-IL-4, differentiate into CD8+ T cells secreting a Th1-like cytokine pattern (IL-2 and IFN gamma). IL-4 induced differentiation into CD8+ T cells secreting Th2 cytokines (IL-4, IL-5, IL-6, and IL-10), whereas anti-IFN gamma suppressed the development of CD8+ cells secreting IFN gamma. Clones of IL-4- or IFN gamma-producing CD8+ T cells were relatively stable, as IL-4 or IFN gamma did not cause interconversion of committed CD8+ T cells. Both CD8+ subsets were cytotoxic, failed to provide cognate help for B cell antibody production, and remained CD4-, CD8 alpha+ CD8 beta+. We propose the names TC1 and TC2 for cytotoxic CD8+ T cells secreting Th1-like and Th2-like cytokines, respectively.

Differentiation of subsets of CD4+ and CD8+ T cells.

Our knowledge of the cytokine secretion patterns of T cells and other cells is clearly becoming more complex. The T helper 1 (Th1) and Th2 patterns may represent the extremes of a spectrum of cytokine regulatory patterns controlled by several cell types. CD8+ T cells can also secrete either Th1-like or Th2-like cytokine patterns, and they can contribute to bystander B cell activation. Interactions occur between immune cytokine regulatory networks and other systems, and pregnancy and responses against infection can profoundly influence each other.

IL-10 inhibits cytokine production, vascular leakage, and swelling during T helper 1 cell-induced delayed-type hypersensitivity.

The Th1 group of cytokines (IL-2, IFN-gamma, and lymphotoxin (LT)) is usually associated with delayed-type hypersensitivity (DTH), whereas Th2 cytokines (IL-4, IL-5, IL-6, and IL-10) often accompany Ab production. Strong DTH and Ab responses are often mutually exclusive, possibly because of negative regulation between the cytokine patterns. In vitro, IL-10 inhibits the production of cytokines by Th1 cells, and inflammatory mediators by monocytes/macrophages, suggesting that IL-10 may be a negative regulator for DTH. We have investigated the effect of systemic IL-10 on the DTH reaction (nonindurated, with edema and granulocytic infiltration) induced by injection of Th1, clones into mouse footpads. Mammalian mouse rIL-10 was purified to > 90% homogeneity. The apparent in vivo half-life of i.p. injected IL-10 was approximately 2 h. Systemically administered IL-10 inhibited the 24-h footpad swelling induced by allo- and Ag-specific Th1 clones to a variable degree, with the strongest effect coinciding with peak DTH swelling. IL-10 also inhibited footpad swelling induced by a secondary challenge in SRBC-primed mice by 25 to 40%. Inhibition of Th1-induced swelling was accompanied by a similar inhibition of vascular permeability, as measured by leakage of Evans blue. Levels of IL-2, IL-6, IL-10, IFN-gamma, and TNF-alpha/LT were elevated in footpads undergoing a DTH reaction. IL-10 treatment reduced the levels of Th1 cytokines (IL-2, IFN-gamma, and TNF-alpha/LT) as well as IL-6, that was probably synthesized by other cells as a result of Th1 activation. The correlation between the inhibition of footpad swelling and cytokine production suggested that the effect of IL-10 on DTH may be mediated through suppression of cytokine synthesis.